Contributors

Glen N. Barber* Jean Dubuisson*

Room 511 Papanicolau Building CNRS-UPR2511
1550 NW 10th Ave [M710] Institut de Biologie de Lille - Institut Pasteur
University of Miami School of Medicine de Lille
Miami Lille
FL 33136 France
USA
David N. Frick*
Keril J. Blight* Department of Biochemistry and Molecular
Department of Molecular Microbiology Biology
Center for Infectious Disease Research New York Medical College
Washington University School of Medicine Valhalla
660 South Euclid Ave NY 10595
Campus Box 8230 USA
St. Louis
MO 63110-1093 Jeffrey S. Glenn*
USA Division of Gastroenterology and Hepatology
Stanford University School of Medicine
D. Spencer Carney CCSR Building
Department of Digestive and Liver Diseases Room 3115
University of Texas Southwestern Medical 269 Campus Drive
Center Palo Alto
5323 Harry Hines Blvd. CA 94305-5187
Dallas TX USA
75390-9048
USA Anne Goffard
CNRS-UPR2511
Stéphane Chevaliez Institut de Biologie de Lille - Institut Pasteur
Department of Virology de Lille
INSERM U635 Lille
Henri Mondor Hospital France
University of Paris 12
Créteil David R. Gretch*
France Laboratory Medicine
Virology
Linda B. Couto Box 359690
Benitec LLC 325 9th Ave
2375 Garcia Ave Seattle
Mountain View WA 98104-2499
CA 94043 USA
USA
Xiao-Song He*
Michael Gale Jr.* VA Medical Center 154C
Department of Microbiology Building 101
University of Texas Room C4-171
Southwestern Medical Center 3801 Miranda Ave. Palo Alto
5323 Harry Hines Blvd. CA94304
Dallas TX USA
75390-9048
USA

v
Yupeng He Chao Lin*
Abbott Laboratories Department of Infectious Diseases
Abbott Park Vertex Pharmaceuticals Incorporated
IL 60064 130 Waverly Street
USA Cambridge
Massachusetts 02139
Cheng Kao USA
Department of Biochemistry and Biophysics
Texas A&M University Jaisri R. Lingappa
College Station Department of Pathobiology
TX 77843 University of Washington
USA Seattle
WA
Takanobu Kato USA
Liver Diseases Branch
NIDDK Ayaz M. Majid
National Institute of Health Department of Microbiology and Immunology
Bethesda University of Miami School of Medicine and
Maryland 20892 Sylvester Comprenhensive Cancer Center
USA Miami
Florida
Kevin C. Klein USA
Department of Pathobiology
University of Washington Tatsuo Miyamura
Seattle Department of Virology II
WA National Institute of Infectious Diseases
USA 1-23-1 Toyama
Shinjuku-ku
Alexander A. Kolykhalov* Tokyo 162-8640
Benitec LLC Japan
2375 Garcia Ave
Mountain View Elizabeth A. Norgard
CA 94043 Department of Molecular Microbiology
USA Center for Infectious Disease Research
Washington University School of Medicine
Michael M. C. Lai* 660 South Euclid Ave
University of Southern California Campus Box 8230
Keck School of Medicine St. Louis
2011 Zonal Avenue MO 63110-1093
Los Angeles USA
CA 90033
USA Arnim Pause*
McGill Cancer Center and Department of
Muriel Lavie Biochemistry
CNRS-UPR2511 McGill University
Institut de Biologie de Lille Montreal
Institut Pasteur de Lille Quebec
Lille H3G 1Y6
France Canada

vi
Jean-Michel Pawlotsky* Ella H. Sklan
Department of Virology Division of Gastroenterology and Hepatology
INSERM U635 Stanford University School of Medicine
Henri Mondor Hospital CCSR Building
University of Paris 12 Room 3115
Créteil 269 Campus Drive
France Palo Alto
CA 94305-5187
Stephen J. Polyak* USA
Department of Laboratory Medicine
Virology Division Kirk A. Staschke
Box 359690 Lilly Research Laboratories
325 9th Ave. Indianapolis
Seattle IN 46285
WA 98104-2499 USA
USA
Seng-Lai Tan*
C. T. Ranjith-Kumar* Lilly Research Laboratories
Department of Biochemistry and Biophysics Indianapolis
Texas A&M University IN 46285
College Station USA
TX 77843
USA Takaji Wakita*
Department of Microbiology
Stephanie T. Shi Tokyo Metropolitan Institute for Neuroscience
Department of Virology 2-6 Musashidai
Pfizer Inc. Fuchu
San Diego Tokyo 183-8526
CA 92121 Japan
USA
Sarah Welbourn
Ikuo Shoji McGill Cancer Center and Department of
Department of Virology II Biochemistry
National Institute of Infectious Diseases McGill University
1-23-1 Toyama Montreal
Shinjuku-ku Quebec
Tokyo 162-8640 H3G 1Y6
Japan Canada

* Corresponding author

vii
 Preface

Chronic hepatitis C is a serious public health problem and a disease burden in

many parts of the world. The discovery of the causative agent, hepatitis C virus
(HCV), in 1989 has initiated an almost unparalleled research activity in academic
and pharmaceutical-industry laboratories over the ensuing years. This book aims to
provide a state-of-the-art account of recent advances in the molecular and cellular
biology, immunology and pathogenesis of HCV. It also aspires to discuss new
strategies as well as outstanding issues for future research.

Hepatitis C has been dubbed the "silent epidemic" because it is generally

asymptomatic for decades after infection; its victims often are unaware of the
infection until it is too late for therapy. What is the genetic makeup and molecular
features that make HCV such a "silent" yet deadly assassin? This question, in fact,
is the premise by which this monograph was prepared – it was an attempt to decode
the secrets of HCV, one viral gene at a time. To that end, we assembled a team of
highly regarded experts from different disciplines who have prepared 16 chapters
on various aspects of HCV, including the HCV genome and the role(s) of each
viral gene product within the context of the viral life cycle, host interactions, and
regulation of host antiviral defense and adaptive immunity.

This book can be divided into six main sections. The Introduction sets the stage by
providing an overview of the history and the significant hallmarks in the discovery,
diagnosis and initial treatments of HCV infection. In the first section, the authors
provide an overview of our present understanding of the HCV genome, the structure
and replication of these viruses (Chapter 1) and the role of the non-coding regions
of HCV in regulating HCV gene expression and RNA replication (Chapter 2).
The next two sections include in-depth reviews of the structural (Chapters 3 and
4) and nonstructural (Chapters 5-10) proteins of HCV. A major drawback in the
past has been the lack of a robust cell-culture and small-animal model system for
HCV infection and replication. However, substantial scientific progress has been
made in recent years (Chapters 11-12). Armed with these tools, we are beginning
to dissect the molecular mechanisms by which the virus disrupts the host innate
and adaptive immune response (Chapters 13 and 14), yielding novel insights into
the pathogenicity of HCV.

The final section covers the development of infectious HCV-like particle systems
(Chapter 15) and the recently developed robust in vitro HCV infection systems
based on the JFH-1strain (Chapter 16), which should greatly expedite our study
of the full viral life cycle, and our efforts to construct anti-viral strategies and
to develop effective immunization strategies with prophylactic and therapeutic

ix
potential. Needless to say, this is the Holy Grail of HCV research considering that
there is no vaccine available and current treatments fail in about half of HCV-
infected patients.

In the meantime, biotechnology and pharmaceutical companies are making

exciting progress in discovery and development of new drugs for HCV therapeutic
intervention. These have been the subject of many excellent recent reviews and thus
will not be covered in this book. Although it is likely to be several years before any
new drug candidate will be available as an anti-HCV agent, the clinical pipeline for
hepatitis C is starting to show promise for safer and more effective therapies.

Seng-Lai Tan, Ph.D.

Indianapolis, May 2006

x
 Introduction

Introduction
David R. Gretch

When the term emerging infectious diseases is loosely applied, then chronic hepatitis
C is recognized as one of the most important new diseases afflicting man. The term
paradigm is useful when describing this disease, since the discovery, diagnosis
and initial treatments of hepatitis C virus infection are all perfect examples of the
increasing impact molecular biology is currently having on disease management
throughout the globe. The discovery of HCV in the late 1980s occurred without the
aid of conventional tissue culture or classical virological methods other than the
essential reliance of the chimpanzee model for propagation and initial definition
of the infectious agent as an enveloped RNA virus. Reverse transcription and PCR
amplification of a subgenomic fraction of the HCV genome not only lead to the
initial genetic characterization of HCV as a putative member of the Pestivirus family.
It also paved the way for development of the first diagnostic test, an enzyme linked
immunoassay that utilized recombinant HCV protein fragments to capture HCV
antibodies from patient serum and thus provide serological evidence of infection.
This critical step was a major accomplishment for molecular medicine since it
provided the first opportunity to positively identify individuals with this highly
prevalent yet clinically silent disease.

Even though it was well established from epidemiological studies that non-A,
non-B (NANB) hepatitis was efficiently transmitted by blood transfusion, and that
screening blood products for anti-hepatitis B core antibody and ALT significantly
reduced the incidence of post-transfusion NANB hepatitis, development of the first
generation HCV antibody screening assay had an impact far greater than many
medical scientists in the field had anticipated. Results of early studies indicated
that up to 10% of all units of blood transfused in the U.S. prior to the discovery of
HCV had lead to transmission of the infectious agent to recipients, accounting for
the vast majority of cases of post-transfusion NANB hepatitis, a fact that may have
been as surprising as it was fortuitous. However, this was not the whole iceberg;
world wide population-based studies revealed a global seroprevalence of well over
100 million individuals, with current estimates being frequently quoted as 170
million HCV infections today. Initial studies reported that approximately 40% of
HCV infections in the U.S. were "community acquired", with no known risk factors
for acquisition. Subsequent epidemiological studies have suggested that many of

1
Gretch

these cases were actually associated with the most important risk factor for HCV
acquisition today, namely intravenous drug use. Such studies have also led to the
identification of other previously unknown risk factors, so the term community
acquired hepatitis C is no longer in vogue. Thus, cloning of a portion of the HCV
RNA genome and development of an effective diagnostic test for HCV antibodies
unveiled the insidious disease that is so heavily researched today; this would never
have occurred without the use of molecular tools.

A second major accomplishment of molecular medicine was development and

standardization of tests that efficiently detect and characterize HCV nucleic
acids in patient blood. Use of the reverse transcription polymerase chain reaction
(RT-PCR) assay in epidemiological studies revealed that of all patients acquiring
post-transfusion hepatitis C, over 80% became chronically infected with persistent
viremia for decades if not for life. Being able to define HCV viremia in a patient
with a risk factor for infection or an asymptomatic individual with serological
evidence of exposure to HCV has become a hallmark tool for hepatitis C
management in the clinical setting from several perspectives. Confirmation of
viremia equals confirmation of active disease. Since most patients with hepatitis
C are asymptomatic, a large percentage has normal ALT levels in the blood, and
up to 20% of infections spontaneously resolve, knowing HCV infection status is
critical for defining subsequent management. Determining whether the disease is
mild, moderate or severe still requires a liver biopsy unless the patient has clinical
evidence of liver decompensation.

The ability to detect, quantify and genetically characterize HCV RNA in patients
had an irreplaceable impact on our understanding of hepatitis C disease long
before the molecular studies described in the following chapters began to unravel
the complex mysteries associated with this truly unique virus-host relationship.
Studies of HCV molecular epidemiology indicated that six distinct genotypes have
evolved over centuries throughout the world. From clinical studies we learned
that HCV persistently replicates in humans for decades, maintaining remarkably
constant serum titers that often exceed 1 million viral genomes per milliliter of
serum. Pharmacodynamic studies indicated that the HCV production rate exceeds
one trillion new virions per day in the face of active immune responses, which is
remarkable because this level of virus production is often without overt detriment
to the infected host. However, HCV continuously evolves within the host as a
pool of genetic variants termed viral quasispecies, presumably as an adaptation to
host pressure. How host pressures shape these viral quasispecies without causing
significant perturbations in HCV RNA titers is also a mystery, as is the mechanistic
relationship between host pressure, viral evolution and disease progression. Again,
development of HCV nucleic acid-based assays was an essential contribution of
molecular medicine in terms of furthering our understanding of the fundamental

2
 Introduction

virology of HCV infection in humans, and defining the mysteries of viral

pathogenesis that may never be approachable for study by in vitro models.

An additional point to touch on with regards to the contribution of molecular

medicine to hepatitis C disease pertains to recombinant human interferon, a drug
that was engineered from the human genome many decades ago as new wonder
drug for the treatment of cancer. Although the utility of interferon in treating cancer
should not be understated, it was the astute observations of clinical investigators in
the pre-hepatitis C era that interferon lead to normalization of serum ALT levels in
about 50% of subjects treated for NANB hepatitis, a remarkable finding even before
the discovery of the etiological agent. Long-term studies indicated that although
many of these patients relapsed after completion of interferon treatment, several
patients continue to have durable and sustained responses with long-term clearance
of HCV RNA from blood. Thus, it is not unreasonable to assume that exogenous
interferon alone can lead to a cure of this highly efficient virus from the infected
host. We now know that HCV genotype and viral load are independent predictors
of response to interferon, and other viral factors have also been implicated in
influencing treatment outcome.

Molecular testing also played an essential role in the optimization of therapy for
hepatitis C. Sentinel studies of HCV RNA dynamics following acute interferon
dosing not only revealed a rapid dose response effect that was not previously
recognized, they also lead directly to the understanding that thrice weekly dosing
of interferon was not optimal. At the same time came the serendipitous discovery
that the more traditional antiviral agent ribavirin potentiates long-term response to
interferon by greatly reducing post-therapy relapse. The end result: the development
and licensing of a much more effective combination therapy for hepatitis C,
including a pegylated interferon compound with extended half-life, plus ribavirin.
Today combination therapy gives clinicians the ability to achieve sustained clearance
of HCV and subsequent improvements in liver disease in over 50% of their treated
patients. This is an outstanding accomplishment when one considers the relatively
poor prognosis for durable sustained remissions in other insidious chronic diseases
in humans. Optimization of therapy through traditional clinical trial research
without the use of molecular analysis of HCV RNA may never have lead to such
a dramatic improvement in hepatitis C treatment outcome. It is at this point that
present research takes over with the clear goal of developing new therapies capable
of improving long-term response rates in those patients who remain resistant to the
best available conventional therapies.

It is this problem combined with the perplexing molecular clinical biology of chronic
hepatitis C that has fueled the enormous surge in basic research that is the topic
of the following chapters. Over a decade of research in the chimpanzee model has

3
Gretch

provided much relevant information with respect to HCV infection and immunity
in the host, and small animal models have been developed which should become
important tools for further characterizing HCV biology in the near future. Aside
from the ever growing body of knowledge related to basic HCV virology, several
key interactions between HCV proteins and the host cell regulatory pathways have
now been described, including some which have exciting potential in terms of
designing new approaches to therapy. Development of the HCV replicon provided
for the first time a highly efficient system for studying HCV protein function during
viral replication and the effects of experimental drugs on specific aspects of the
viral life cycle. However, one important limitation of the HCV replicon is the
fact that infectious virus is not produced; thus it falls short of the ideal. Although
the lack of a robust tissue culture system has been a major impediment to HCV
research in the past, productive infection of culture cells by a unique HCV isolate
has very recently been reported. It is the hope of investigators that this system will
now provide the opportunity to study for the first time several essential steps in
the HCV life cycle. However, it is also essential that more flexible and even more
robust infection models be continuously developed.

In summary, both the intensity and breadth of HCV research are growing at a
remarkable pace, and exciting new discoveries are becoming almost commonplace
in the literature. The following chapters were written to provide in-depth reviews
of several of the most critical areas of HCV molecular research today. However, it
is the goal of this Introduction to remind readers and investigators that hepatitis C
disease is highly complex and very likely involves multiple poorly defined viral-
host interactions that still cannot be and may never be recapitulated in any animal
model or in vitro system. For this reason, molecular research into other Pestivirus
animal disease models should be pursued with renewed vigor. Finally, continuous
research in the human disease model is essential for defining the most important
questions for in vitro study, as is the continuous development of new molecular
tools for dissecting the intriguing biopathogenesis of chronic hepatitis C in man.
Just as the progress on this disease to date has been phenomenal, so too will be the
future progress in furthering our understanding of HCV infection, replication, and
molecular biology, and in improving the treatment of hepatitis C. The present state
of progress and unanswered questions currently facing molecular investigators are
both very well summarized in the following chapters. As for molecular medicine,
hepatitis C may long remain the essential paradigm of how new technologies can
impact in a very real manner existing problems afflicting man.

4
 Genome and Life Cycle

Chapter 1

HCV Genome and Life Cycle

Stéphane Chevaliez and Jean-Michel Pawlotsky

ABSTRACT
Hepatitis C virus (HCV) infection afflicts more than 170 million people worldwide,
with the great majority of patients with acute hepatitis C developing chronic HCV
infection. It can ultimately result in liver cirrhosis, hepatic failure or hepatocellular
carcinoma, which are responsible for hundreds of thousands of deaths each year.
Despite the discovery of HCV over 15 years ago, our knowledge of the HCV
lifecycle has been limited by our inability to grow the virus in cell culture, as
well as by the lack of small-animal models of HCV infection. Nevertheless,
data accumulated through the use of multiple in vitro and in vivo study systems
have provided a general picture of the biology of HCV, although sometimes with
contradictory results. Herein, we summarize our current understanding of the HCV
genome and how its structure and encoded gene products, in a complex interplay
with host cell factors, might orchestrate a productive viral lifecycle while evading
the scrutiny of the host immune system. The recently developed robust in vitro
HCV infection systems should help fill in some of the gaps in understanding the
HCV lifecycle in the next few years.

HCV GENOME ORGANIZATION AND FUNCTION

The Flaviviridae family is divided into three genera: flavivirus, pestivirus, and
hepacivirus. Flaviviruses include yellow fever virus, dengue fever virus, Japanese
encephalitis virus, and Tick-borne encephalitis virus. Pestiviruses include bovine
viral diarrhea virus, classical swine fever virus and Border disease virus. HCV, with
at least 6 genotypes and numerous subtypes, is a member of the hepacivirus genus,
which includes tamarin virus and GB virus B (GBV-B) and is closely related to
human virus GB virus C (GBV-C) (Lindenbach and Rice, 2001).

The members of the Flaviviridae family share a number of basic structural and
virological characteristics. They are all enveloped in a lipid bilayer in which two or
more envelope proteins (E) are anchored. The envelope surrounds the nucleocapsid,
which is composed of multiple copies of a small basic protein (core or C), and
contains the RNA genome. The Flaviviridae genome is a positive-strand RNA
molecule ranging in size from 9.6 to 12.3 thousand nucleotides (nt), with an open
reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more.

5
Chevaliez and Pawlotsky

The structural proteins are encoded by the N-terminal part of the ORF, whereas
the remaining portion of the ORF codes for the nonstructural proteins (Fig. 1).
Sequence motif-conserved RNA protease-helicase and RNA-dependant RNA
polymerase (RdRp) are found at similar locations in the polyproteins of all of the
Flaviviridae (Miller and Purcell, 1990). In addition, all Flaviviridae share similar
polyprotein hydropathic profile, with flaviviruses and hepaciviruses being closer
to each other than to pestiviruses (Choo et al., 1991). The ORF is flanked in 5' and
3' by untranslated regions (UTR) of 95-555 and 114-624 nt in length, respectively,
which play an important role in polyprotein translation and RNA replication (Fig.
1) (Thurner et al., 2004).

Flaviviridae bind to one or more cellular receptors organized as a receptor

complex and appear to trigger receptor-mediated endocytosis. Fusion of the virion
envelope with cellular membranes delivers the nucleocapsid to the cytoplasm. After
decapsidation, translation of the viral genome occurs in the cytoplasm, leading to
the production of a precursor polyprotein, which is then cleaved by both cellular
and viral proteases into structural and nonstructural proteins. Replication of the
viral genome is carried out by the viral replication complex which is associated
with cellular membranes and resistant to actinomycin D. Viral replication occurs in
the cytoplasm via the synthesis of full-length negative-strand RNA intermediates.
Progeny virions are assembled from cytoplasmic vesicles formed by budding

Fig. 1. Organization of Flaviviridae genomes. The figure shows, from top to bottom, the genomes of
HCV (hepacivirus), pestivirus, and yellow fever virus (flavivirus). NS: non structural.

6
 Genome and Life Cycle

through intracellular membranes. Finally, mature virions are released into the
extracellular milieu by exocytosis.

Despite the above-mentioned similarities with the members of other Flaviviridae

genera, HCV does exhibit a number of virological, epidemiological as well
as pathophysiological differences. Flavivirus translation is cap-dependent, i.e.
mediated by a type I cap structure located in the 5'UTR (m7GpppAmp), followed by
the conserved AG sequence and a relatively short stretch upstream of the polyprotein
coding region (Brinton and Dispoto, 1988). In contrast, the HCV 5'UTR is not
capped and, like that of pestiviruses and GB viruses, folds into a complex secondary
RNA structure forming, together with a portion of the core-coding domain, an
internal ribosome entry site (IRES) that mediates direct binding of ribosomal
subunits and cellular factors and subsequent translation (see Chapter 2). Whereas
the flavivirus 3' UTR is highly structured, the HCV 3'UTR is relatively short, less
structured and contains a poly-uridyl tract that varies in length.

HCV has a narrow host specificity and tissue tropism. HCV is transmitted
exclusively through direct blood-to-blood contacts between humans. Flaviviruses
are principally vectored by mosquitoes or ticks and can infect a broad range of
vertebrate animals, with humans being a dead-end host that does not participate
in the perpetuation of virus transmission. No known pestivirus can infect humans
and no known insect vector has been identified. Infections caused by flaviviruses
are acute-limited in vertebrate animals, whereas HCV has a high chronicity rate in
humans (50%-80%, depending on the age at infection). Strong and adapted humoral
and cellular immune responses have been shown to be involved in flavivirus and
pestivirus infection recovery and protection. However, HCV infection induces an
immune response that fails to prevent chronicity in most cases and does not confer
protection against reinfection with homologous and heterologous strains in the
chimpanzee model (Farci et al., 1997).

HCV GENOME STRUCTURE AND ORGANIZATION

The structural organization of HCV genome is schematically depicted in Fig. 2.

5' UNTRANSLATED REGION

The HCV 5'UTR contains 341 nt located upstream of the ORF translation initiation
codon. It is the most conserved region of the genome (nt sequence identity is 60%
with GBV-B and approximately 50% with pestiviruses (Choo et al., 1991; Han et
al., 1991). The 5'UTR contains four highly structured domains, numbered I to IV,
containing numerous stem-loops and a pseudoknot (Brown et al., 1992; Wang et
al., 1995). Domains II, III and IV together with the first 12 to 30 nt of the core-
coding region constitute the IRES (Honda et al., 1996). Structural characterization
by electron microscopy (EM) indicated that domains II, III and IV form distinct

7
Chevaliez and Pawlotsky

Fig. 2. HCV genome organization (top) and polyprotein processing (bottom). The 5'UTR consists
of four highly structured domains and contains the IRES. The 3'UTR consists of stable stem-loop
structures and an internal poly(U)-poly(U/C) tract. The central 9.6-kb ORF codes for a polyprotein of
slightly more than 3000 aa depending on the HCV genotype. S and NS correspond to regions coding
for structural and nonstructural proteins, respectively. The polyprotein processing and the location of
the 10 HCV proteins relative to the ER membrane are schematically represented. Scissors indicate
ER signal peptidase cleavage sites; cyclic arrow, autocatalytic cleavage of the NS2-NS3 junction;
black arrows, NS3-NS4A protease complex cleavage sites; intramembranous arrow, cleavage by the
signal peptide peptidase. The transmembrane domains of E1 and E2 are shown after signal-peptidase
cleavage and reorientation of the respective C-terminus hydrophobic stretches (dotted rectangles).
Spots denote glycosylation sites of the E1 and E2 envelope proteins. Reproduced from Penin et al.,
2004b with permission.

regions within the molecule, with a flexible hinge between domains II and III
(Beales et al., 2001). The HCV IRES has the capacity to form a stable pre-initiation
complex by directly binding the 40S ribosomal subunit without the need of canonical
translation initiation factors, an event that likely constitutes the first step of HCV
polyprotein translation.

Several reports suggested a tissue compartmentalization of IRES sequences (Laskus

et al., 2000; Lerat et al., 2000; Nakajima et al., 1996; Shimizu et al., 1997). Infection
of lymphoid cell lines with HCV genotype 1a H77 strain led to the selection of a
quasispecies with nucleotide substitutions within the 5'UTR relative to the inoculum
that conferred a 2- to 2.5-fold increase in translation efficiency in human lymphoid
cell lines relative to monocyte, granulocyte or monocyte cell lines (Lerat et al.,
2000). Furthermore, different translation efficiencies of HCV quasispecies variants
isolated from different cell types in the same patient were observed, suggesting cell
type-specific IRES interactions with cellular factors may also modulate polyprotein
translation (Forton et al., 2004; Laporte et al., 2000; Lerat et al., 2000).

8
 Genome and Life Cycle

3' UNTRANLATED REGION

The 3'UTR contains approximately 225 nt. It is organized in three regions including,
from 5' to 3', a variable region of approximately 30-40 nt, a long poly(U)-poly(U/
UC) tract, and a highly conserved 3'-terminal stretch of 98 nt (3'X region) that
includes three stem-loop structures SL1, SL2 and SL3 (Kolykhalov et al., 1996;
Tanaka et al., 1995; Tanaka et al., 1996). The 3'UTR interacts with the NS5B
RdRp and with two of the four stable stem-loop structures located at the 3' end of
the NS5B-coding sequence (Cheng et al., 1999; Lee et al., 2004). The 3'X region
and the 52 upstream nt of the poly(U/C) tract were found to be essential for RNA
replication, whereas the remaining sequence of the 3'UTR appears to enhance viral
replication (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon,
2003a; Yi and Lemon, 2003b).

CHARACTERISTICS AND FUNCTIONS OF HCV PROTEINS

The HCV ORF contains 9024 to 9111 nt depending on the genotype. The ORF
encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and
E2), a small protein, p7, whose function has not yet been definitively defined, 6
nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), and the
so-called "F" protein which results from a frameshift in the core coding region
(Fig. 2; Table 1). The characteristics and functions of HCV proteins are extensively

Table 1. HCV proteins and their functions in the viral life cycle. Adapted from Bartenschlager et
al., 2004.
HCV protein Function Apparent molecular weight
(kDa)
Core Nucleocapsid 23 (precursor)
21 (mature)
F/ARFa-protein ? 16-17
E1 Envelope 33-35
Fusion domain?
E2 Envelope 70-72
Receptor binding
Fusion domain?
p7 Calcium ion channel (viroporin) 7
NS2 NS2-3 autoprotease 21-23
NS3 Component of NS2-3 and NS3-4A proteinases 69
NTPase/helicase
NS4A NS3-4A proteinase cofactor 6
NS4B Membranous web induction 27
NS5A RNA replication by formation of replication 56 (basal form)
complexes 58 (hyperphosphorylated
form)
NS5B RNA-dependant RNA polymerase 68
a Frameshift/ alternate reading frame

9
Chevaliez and Pawlotsky

described elsewhere in this book. Here, we provide a brief overview of the viral
gene products and their roles in the HCV lifecycle.

STRUCTURAL PROTEINS

CORE PROTEIN
The HCV core protein is a highly basic, RNA-binding protein, which presumably
forms the viral capsid (see Chapter 3). The HCV core protein is released as a 191
aa precursor of 23-kDa (P23). Although proteins of various sizes (17 to 23 kDa)
were detectable, the 21-kDa core protein (P21) appeared to be the predominant form
(Yasui et al., 1998). The core protein contains three distinct predicted domains : an
N-terminal hydrophilic domain of 120 aa (domain D1), a C-terminal hydrophobic
domain of about 50 aa (domain D2), and the last 20 or so aa that serve as a signal
peptide for the downstream envelope protein E1 (Grakoui et al., 1993c; Harada et
al., 1991; Santolini et al., 1994). Domain D1 contains numerous positive charges. It
is principally involved in RNA binding and nuclear localization, as suggested by the
presence of three predicted nuclear localization signals (NLS) (Chang et al., 1994;
Suzuki et al., 1995; Suzuki et al., 2005). Domain D2 is responsible for core protein
association with endoplasmic reticulum (ER) membranes, outer mitochondria
membranes and lipid droplets (Schwer et al., 2004; Suzuki et al., 2005).

Both membrane-bound and membrane-free core proteins appear to exist as dimeric

or multimeric forms. When expressed in various in vitro systems, including cell-free
or mammalian, bacterial, insect or yeast cell culture models, the HCV core protein
can form nucleocapsid-like particles (NLPs) (Baumert et al., 1998; Blanchard et al.,
2002; Blanchard et al., 2003; Dash et al., 1997; Ezelle et al., 2002; Iacovacci et al.,
1997; Klein et al., 2004; Mizuno et al., 1995; Pietschmann et al., 2002; Serafino et
al., 1997; Shimizu et al., 1996). The region between aa 82 and 102 of hydrophilic
domain D1 contains a tryptophan-rich sequence and has been suggested to allow
the P21 core protein to interact with itself, a property not borne by the precursor
P23 (Nolandt et al., 1997). On the other hand, the 75 N-terminal residues of the
core protein appear sufficient for NLP assembly in a bacterial system (Majeau et
al., 2004). Recently, two clusters of basic residues located in the 68 N-terminal nt
were shown to play a critical role in capsid assembly in a cell-free system, whereas
the region between aa 82 and 102 did not play a major role (Klein et al., 2005). The
critical residues for capsid assembly remain to be precisely identified.

In addition to its role in viral capsid formation, the core protein has been suggested
to directly interact with a number of cellular proteins and pathways that may be
important in the viral lifecycle (McLauchlan, 2000). The HCV core protein has
pro- and anti-apoptotic functions (Chou et al., 2005; Kountouras et al., 2003; Meyer
et al., 2005), stimulates hepatocyte growth in Huh-7 cell line by transcriptional
upregulation of growth-related genes (Fukutomi et al., 2005), and has been

10
 Genome and Life Cycle

implicated in tissue injury and fibrosis progression (Nunez et al., 2004). The HCV
core protein could also regulate the activity of cellular genes, including c-myc and
c-fos, and alter the transcription of other viral promoters (Ray et al., 1995; Shih et
al., 1993). It induces hepatocellular carcinoma when expressed in transgenic mice
(Moriya et al., 1998; Moriya et al., 1997). It could also induce the formation of
lipid droplets and may play a direct role in steatosis formation (Barba et al., 1997;
Moriya et al., 1998; Moriya et al., 1997).

E1 AND E2 ENVELOPE GLYCOPROTEINS

The two envelope glycoproteins, E1 and E2, are essential components of the
HCV virion envelope and necessary for viral entry and fusion (Bartosch et al.,
2003a; Nielsen et al., 2004) (see Chapter 4). E1 and E2 have molecular weights
of 33-35 and 70-72 kDa, respectively, and assemble as noncovalent heterodimers
(Deleersnyder et al., 1997). E1 and E2 are type I transmembrane glycoproteins, with
N-terminal ectodomains of 160 and 334 aa, respectively, and a short C-terminal
transmembrane domain of approximately 30 aa. The E1 and E2 transmembrane
domains are composed of two stretches of hydrophobic aa separated by a short
polar region containing fully conserved charged residues. They have numerous
functions, including membrane anchoring, ER localization and heterodimer
assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). The ectodomains of
E1 and E2 contain numerous proline and cysteine residues, but intramolecular
disulfide bonds have not been observed (Matsuura et al., 1994). E1 and E2 are
highly glycosylated, containing up to 5 and 11 glycosylation sites, respectively. In
addition, E2 contains hypervariable regions with aa sequences differing up to 80%
between HCV genotypes and between subtypes of the same genotype (Weiner et
al., 1991). Hypervariable region 1 (HVR1) contains 27 aa and is a major (but not
the only) HCV neutralizing epitope (Farci et al., 1996; Zibert et al., 1997). Despite
the HVR1 sequence variability, the physicochemical properties of the residues at
each position and the overall conformation of HVR1 are highly conserved among
all known HCV genotypes, suggesting an important role in the virus lifecycle
(Penin et al., 2001).

E2 plays a crucial role in the early steps of infection. Viral attachment is thought
to be initiated via E2 interaction with one or several components of the receptor
complex (Flint and McKeating, 2000; Rosa et al., 1996). Because HVR1 is a basic
region with positively charged residues located at specific sequence positions, it can
theoretically interact with negatively charged molecules at the cell surface. This
interaction could play a role in host cell recognition and attachment, as well as in
cell or tissue compartmentalization (Barth et al., 2003; Bartosch et al., 2003b). In
addition, it was recently shown that human serum facilitated infection of Huh7
cells by HCV pseudoparticles, apparently mediated through an interplay between
serum high-density lipoproteins (HDL), HVR1 and the scavenger receptor B type
I (SR-BI) (Bartosch et al., 2005; Voisset et al., 2005). Less is known about E1, but

11
Chevaliez and Pawlotsky

it is thought to be involved in intra-cytoplasmic virus-membrane fusion (Flint and

McKeating, 2000; Rosa et al., 1996).

FRAMESHIFT PROTEIN
The F (frameshift) protein or ARFP (alternate reading frame protein) is generated
as a result of a -2/+1 ribosomal frameshift in the N-terminal core-encoding region
of the HCV polyprotein. Antibodies to peptides from the F protein were detected
in chronically infected patients, suggesting that the protein is produced during
infection (Walewski et al., 2001). However, the exact translational mechanisms
governing the frequency and yield of the F protein during the various phases of
HCV infection are completely unknown. Thus, the role of F protein in the HCV
lifecycle remains enigmatic but it was proposed to be involved in viral persistence
(Baril and Brakier-Gingras, 2005).

NONSTRUCTURAL PROTEINS

P7
p7 is a small, 63 aa polypeptide, that has been shown to be an integral membrane
protein (Carrere-Kremer et al., 2002). It comprises two transmembrane domains
organized in α-helices, connected by a cytoplasmic loop. p7 appears to be essential,
because mutations or deletions in its cytoplasmic loop suppressed infectivity of
intra-liver transfection of HCV cDNA in chimpanzees (Sakai et al., 2003). In vitro
studies suggested that p7 belongs to the viroporin family and could act as a calcium
ion channel (Gonzalez and Carrasco, 2003). However, these results remain to be
confirmed in vivo.

NS2
NS2 is a non-glycosylated transmembrane protein of 21-23 kDa (see Chapter 5). It
contains two internal signal sequences at aa positions 839-883 and 928-960, which
are responsible for ER membrane association (Santolini et al., 1995; Yamaga and
Ou, 2002). NS2, together with the amino-terminal domain of the NS3 protein, the
NS2-3 protease, constitutes a zinc-dependent metalloprotease that cleaves the site
between NS2 and NS3 (Grakoui et al., 1993b; Grakoui et al., 1993c; Hijikata et al.,
1993). NS2 is a short-lived protein that looses its protease activity after self-cleavage
from NS3 and is degraded by the proteasome in a phosphorylation-dependent
manner by means of protein kinase casein kinase 2 (Franck et al., 2005). In addition
to its protease activity, NS2 could interact with host cell proteins, such as the liver-
specific pro-apoptotic cell death-inducing DFF45-like effector (CIDE-B), and affect
reporter genes controlled by liver and non-liver-specific promoters and enhancers
(Dumoulin et al., 2003; Erdtmann et al., 2003). However, the consequences of such
interactions within the context of the HCV lifecyle are not clear.

12
 Genome and Life Cycle

NS3-NS4A
NS3 is a multi-functional viral protein containing a serine protease domain in its N-
terminal third and a helicase/NTPase domain in its C-terminal two-thirds. NS4A is
a cofactor of NS3 protease activity. NS3-4A also bears additional properties through
its interaction with host cell pathways and proteins that may be important in the
lifecycle and pathogenesis of infection (see Chapters 6 and 13). Not surprisingly,
the NS3-NS4A protease is one of the most popular viral targets for anti-HCV
therapeutics (Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

NS3-NS4A PROTEASE
The NS3-NS4A protease is essential for the HCV lifecycle. It catalyzes HCV
polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/
NS5B junctions. The 3D structure of the NS3 serine protease domain complexed
with NS4A has been determined (Kim et al., 1996; Love et al., 1996; Yan et
al., 1998). The catalytic triad is formed by residues His 57, Asp 81 and Ser 139
(Bartenschlager et al., 1993; Grakoui et al., 1993a; Tomei et al., 1993). The central
region of NS4A (aa 21–30) acts as a cofactor of NS3 serine protease activity,
allowing its stabilization, localization at the ER membrane as well as cleaveage-
dependent activation, particularly at the NS4B/NS5A junction (Bartenschlager et
al., 1995; Lin et al., 1995; Tanji et al., 1995).

Recently, HCV NS3-NS4A was shown in vitro to antagonize the dsRNA-dependent

interferon regulatory factor 3 (IRF-3) pathway, an important mediator of interferon
induction in response to a viral infection (Foy et al., 2003). NS3-NS4A also appears
to prevent dsRNA signaling via the toll-like receptor 3 upstream of IRF-3 (Li et al.,
2005). One potential mechanism includes a blockade of the intracellular double-
stranded RNA sensor protein (RIG-I) pathway by NS3-NS4A (Sumpter et al.,
2005). Thus, HCV could utilize NS3-4A protease to circumvent the innate immune
response at the early stages of infection. In addition, NS3 was also reported to
induce malignant transformation of NIH3T3 cells (Sakamuro et al., 1995), suppress
actinomycin D-induced apoptosis in murine cell lines (Fujita et al., 1996), and to
be involved in hepatocarcinogenesis events (Borowski et al., 1996; Hassan et al.,
2005), although the exact mechanisms are not clear.

NS3 HELICASE-NTPASE
The NS3 helicase-NTPase domain consisting of the 442 C-terminal aa of the
NS3 protein is a member of the helicase superfamily-2 (see Chapter 7). Its three-
dimentional structure has also been determined (Cho et al., 1998; Kim et al.,
1998; Yao et al., 1997). The NS3 helicase-NTPase has several functions, including
RNA-stimulated NTPase activity, RNA binding, and unwinding of RNA regions of
extensive secondary structure by coupling unwinding and NTP hydrolysis (Gwack
et al., 1997; Tai et al., 1996). During RNA replication, the NS3 helicase has been
suggested to translocate along the nucleic acid substrate by changing protein

13
Chevaliez and Pawlotsky

conformation, utilizing the energy of NTP hydrolysis. A recent study proposed

that the helicase directional movement step is fueled by single-stranded RNA
binding energy, while NTP binding allows for a brief period of random movement
that prepares the helicase for the next cycle (Levin et al., 2005). In addition, NS3
helicase activity appears to be modulated by the NS3 protease domain and the
NS5B RdRp (Zhang et al., 2005).

NS4B
NS4B is an integral membrane protein of 261 aa with an ER or ER-derived
membrane localization (Hugle et al., 2001; Lundin et al., 2003). NS4B is predicted
to harbor at least four transmembrane domains and an N-terminal amphipathic helix
that are responsible for membrane association (Elazar et al., 2004; Hugle et al.,
2001; Lundin et al., 2003). One of the functions of NS4B is to serve as a membrane
anchor for the replication complex (see Chapter 8) (Egger et al., 2002; Elazar et
al., 2004; Gretton et al., 2005). Additional putative properties include inhibition
of cellular syntheses (Florese et al., 2002; Kato et al., 2002), modulation of HCV
NS5B RdRp activity (Piccininni et al., 2002), transformation of NIH3T3 cell lines
(Park et al., 2000), and induction of interleukin 8 (Kadoya et al., 2005).

NS5A
NS5A is a 56-58 kDa phosphorylated zinc-metalloprotein that probably plays an
important role in virus replication and regulation of cellular pathways (see Chapter
9). The N-terminal region of NS5A (aa 1-30) contains an amphipathic α-helix that
is necessary and sufficient for membrane localization in perinuclear membranes
as well as for assembly of the replication complex (Brass et al., 2002; Elazar et
al., 2003; Penin et al., 2004a). Downstream of this motif, the NS5A protein was
predicted to contain three domains, numbered I to III. Domain I, located at the N-
terminus, contains an unconventional zinc-binding motif formed by four cysteine
residues conserved among the hepacivirus and pestivirus genera (Tellinghuisen et
al., 2004). HCV replicon RNA replication was inhibited by mutations in the NS5A
sequence (Elazar et al., 2003; Penin et al., 2004b) and abolished by alterations of the
zinc-binding site (Tellinghuisen et al., 2004). The recently determined 3-D structure
of Domain I suggested the presence of protein, RNA and membrane interaction
sites (Moradpour et al., 2005; Tellinghuisen et al., 2005).

The mechanisms by which NS5A regulate HCV replication are not entirely clear.
NS5A associates with lipid rafts derived from intracellular membranes through
its binding to the C-terminal region of a vesicle-associated membrane-associated
protein of 33 kDa (hVAP-33) (Shi et al., 2003; Tu et al., 1999). This interaction
appears to be crucial for the formation of the HCV replication complex in connection
with lipid rafts (Gao et al., 2004). A recent study in the replicon system proposed a
model in which NS5A hyperphosphorylation disrupts the interaction with hVAP-
33 and negatively regulates viral RNA replication (Evans et al., 2004). Another

14
 Genome and Life Cycle

report suggested that the level of NS5A phosphorylation plays an important role
in the viral lifecycle by regulating a switch from replication to assembly, whereby
hyperphosphorylated forms function to maintain the replication complex in an
assembly-incompetent state (Appel et al., 2005). Furthermore, NS5A can interact
directly with NS5B, but the mechanism by which NS5A modulates the RdRp activity
has not been elucidated (Shimakami et al., 2004). In addition, NS5A was reported
to interact with a geranylgeranylated cellular protein (Wang et al., 2005a). This is
potentially significant considering that assembly of the viral replication complex
has been shown to require geranylgeranylation of one or more host cell proteins
(Ye et al., 2003).

Multiple functions have been assigned to NS5A based on its interactions with
cellular proteins (Tellinghuisen and Rice, 2002) (see Chapter 9). For instance, NS5A
appears to play a role in interferon resistance by binding to and inhibiting PKR, an
antiviral effector of interferon-α (Gale et al., 1998). NS5A also bears transcriptional
activation functions (Pellerin et al., 2004; Polyak et al., 2001) and appears to be
involved in the regulation of cell growth and cellular signaling pathways (Tan and
Katze, 2001; Tellinghuisen and Rice, 2002). However, these observations remain
to be confirmed in vivo.

NS5B RNA-DEPENDENT RNA POLYMERASE

NS5B belongs to a class of membrane proteins termed tail-anchored proteins
(Ivashkina et al., 2002; Schmidt-Mende et al., 2001) (see Chapter 10). Its C-terminal
region (21 residues) forms an α-helical transmembrane domain responsible for
post-translational targeting to the cytosolic side of the ER, where the functional
protein domain is exposed (Moradpour et al., 2004; Schmidt-Mende et al., 2001).
The crystal structure of NS5B revealed that the RdRp has a classical "fingers, palm
and thumb" structure formed by its 530 N-terminal aa (Ago et al., 1999; Bressanelli
et al., 1999; Lesburg et al., 1999). Interactions between the fingers and thumb
subdomains result in a completely encircled catalytic site that ensures synthesis
of positive- and negative-strand HCV RNAs (Lesburg et al., 1999). The RdRp is
another important target for the development of anti-HCV drugs (Di Marco et al.,
2005; Ma et al., 2005; Pawlotsky and McHutchison, 2004; Pawlotsky, 2006).

Interactions between NS5B and cellular components have also been reported.
The C-terminus of NS5B can interact with the N-terminus of hVAP-33, and the
interaction may play an important role in the formation of the HCV replication
complex (Gao et al., 2004; Schmidt-Mende et al., 2001). More recently, NS5B
was reported to bind cyclophilin B, a cellular peptidyl-prolyl cis-trans isomerase
that apparently regulates HCV replication through modulation of the RNA binding
capacity of NS5B (Watashi et al., 2005).

15
Chevaliez and Pawlotsky

THE HCV LIFECYCLE

CELLULAR ATTACHMENT OF HCV VIRIONS AND ENTRY

Many efforts have been made to develop models to identify candidate HCV
receptors and study viral binding and entry into target cells. Various cellular and
in vivo systems utilizing infected blood samples, virus-like particles produced by
expression of structural HCV proteins in insect or mammalian cells, liposomes
containing E1-E2, as well as pseudotype particles have yielded a considerable
amount of data, although they are not always easy to reconcile. Fig. 3 summarizes
the hypothetical HCV lifestyle.

HCV RECEPTORS
Several cell surface molecules have been proposed to mediate HCV binding or
HCV binding and internalization.

CD81
Among all putative HCV receptor molecules, CD81 has been the most extensively
studied (Pileri et al., 1998). Human CD81 (target of antiproliferative antibody 1,
TAPA-1) is a 25-kDa molecule belonging to the tetraspanin or transmembrane 4
superfamily. It is found at the surface of numerous cell types, where it is thought
to assemble as homo- and/or heterodimers by means of a conserved hydrophobic
interface. CD81 contains four hydrophobic transmembrane regions (TM1 to
TM4) and two extracellular loop domains of 28 and 80 aa, respectively: the small
extracellular loop (SEL) and the large extracellular loop (LEL). The LEL is located
between TM3 and TM4. It is composed of five α-helices and contains four cysteine
residues (Kitadokoro et al., 2001). The SEL is needed for optimal surface expression
of the LEL (Masciopinto et al., 2001). The intracellular and transmembrane
domains of CD81 are highly conserved among different species. In contrast, the
LEL is variable, except between humans and chimpanzees, the only two species
permissive to HCV infection (Major et al., 2004; Walker, 1997). The CD81 LEL
has been shown to mediate binding of HCV through its envelope glycoprotein
E2 (Pileri et al., 1998). The integrity of two disulfide bridges is necessary for the
CD81-HCV interaction to occur (Petracca et al., 2000), and the site of interaction
appears to involve CD81 residues 163, 186, 188 and 196 (Flint et al., 1999; Meola
et al., 2000). The E2 domains involved in CD81 binding remain controversial.
Early studies suggested the involvement of aa 480-493 and 544-551 in the truncated
soluble form of E2 (Flint et al., 1999), whereas a more recent study pointed to a
role for two other domains, including aa 613-618 and a second domain spanning
the two HVRs (aa 384-410 and 476-480) (Roccasecca et al., 2003).

Several studies argue that cellular factors other than CD81 are required for HCV
infection. The expression of human CD81 in a CD81-deficient human hepatoma
cell line restored permissiveness to infection with HCV pseudo-particles, but a

16
 Genome and Life Cycle

murine fibroblast cell line expressing human CD81 remained resistant to HCV
entry (Cormier et al., 2004). In addition, expression of human CD81 in transgenic
mice did not confer susceptibility to HCV infection (Masciopinto et al., 2002). It is
possible that the CD81 molecule could act as a post-attachment entry co-receptor
and that other cellular factors act together with CD81 to mediate HCV binding and
entry into hepatocytes (Cormier et al., 2004).

SR-BI
The scavenger receptor B type I (SR-BI) has been proposed as another candidate
receptor for HCV (Scarselli et al., 2002). SR-BI is a 509-aa glycoprotein with
a large extracellular loop anchored to the plasma membrane at both N- and C-
termini by means of transmembrane domains with short cytoplasmic extensions
(Krieger, 2001). SR-BI is a fatty acylated protein located in lipid raft domains. It is
expressed at high levels in hepatocytes and steroidogenic cells (Babitt et al., 1997;
Krieger, 2001). The natural ligand of SR-BI is high density lipoproteins (HDL).
HDLs are internalized through a non-clathrin-dependent endocytosis process that
mediates cholesterol uptake and recycling of HDL apoprotein (Silver et al., 2001).
HCV genotypes 1a and 1b recombinant E2 envelope glycoproteins were shown
to bind HepG2 cells (a human hepatoma cell line that does not express CD81) by
interacting with an 82 kDa glycosylated SR-BI molecule (Scarselli et al., 2002).
Binding appeared to be highly specific: tranfection of rodent cells with human
or tupaia SR-BI (88 % aa identity with human SR-BI) resulted in E2 binding,
whereas neither mouse SR-BI (80 % aa identity) nor the closely related human
scavenger receptor CD36 (60 % aa identity) bound E2. The SR-BI LEL appeared
to be responsible for HCV binding, and HVR1 was recently suggested to be the E2
envelope region involved in the interaction, which was facilitated by serum HDLs
(Bartosch et al., 2003b; Scarselli et al., 2002; Voisset et al., 2005). However, the
fact that antibodies directed against SR-BI resulted only in a partial blockade of
binding suggests that SR-BI is not the only cell surface molecule involved in HCV
binding to hepatocytes (Barth et al., 2005).

DC-SIGN AND L-SIGN

The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin
(DC-SIGN or CD209) and the liver/lymph node-specific intercellular adhesion
molecule-3 (ICAM-3)-grabbing integrin (L-SIGN or CD209L) have been proposed
as tissue-specific capture receptors for HCV present in various cell types that could
play a critical role in viral pathogenesis and tissue tropism (Gardner et al., 2003;
Lozach et al., 2004; Lozach et al., 2003; Pohlmann et al., 2003). DC-SIGN is a
44-kDa homotetrameric type II integral membrane protein with a short amino-
terminal cytoplasmic domain and a carboxy-terminal C-type (calcium-dependent)
lectin domain. DC-SIGN is expressed at a high level on myeloid-lineage dendritic
cells. Its interaction with ICAM-3 activates T cells (Geijtenbeek et al., 2000).
L-SIGN is abundantly expressed at the surface of endothelial cells of the liver

17
Chevaliez and Pawlotsky

and lymph nodes and shares 77% aa sequence identity with DC-SIGN (Bashirova
et al., 2001). A rapid internalization of virus-like particles upon capture of HCV
pseudo-particles by both DC-SIGN and L-SIGN, presumably via E2 binding, was
reported (Ludwig et al., 2004), although this was not observed in another study
(Lozach et al., 2004).

LDL-R
The low-density lipoprotein (LDL) receptor (LDL-R) is an endocytic receptor that
transports lipoproteins, mainly the cholesterol-rich LDLs, into cells through receptor-
mediated endocytosis (Chung and Wasan, 2004). Virus-like particles complexed
with LDLs have been reported to enter into cells via the LDL receptor (Agnello et
al., 1999; Monazahian et al., 1999). In support of this view, binding of low-density
HCV particles recovered from plasma by sucrose gradient sedimentation correlated
with the density of LDL receptors at the surface of MOLT-4 cells and fibroblasts,
and the binding was inhibited by LDL but not by soluble CD81 (Wunschmann et
al., 2000).

ASIALOGLYCOPROTEIN RECEPTOR
The asialoglycoprotein receptor (ASGP-R) has been reported to mediate binding
and internalization of structural HCV proteins (C-E1-E2±p7) expressed in a
baculovirus system. Cotransfection of a non-permissive mouse fibroblast cell
line with cDNAs of both ASGP-R subunits (H1 and H2) restored permissiveness
(Saunier et al., 2003).

GLYCOSAMINOGLYCANS
Conservation of positively charged residues in the N-terminus of E2 is in keeping
with a possible interaction with heparan sulfate proteoglycans (HSPG) (Barth et al.,
2003). E2, in particular its HVR-1, has been shown to bind HSPG with a stronger
affinity than other viral envelope glycoproteins, such as human herpes virus 8 or
dengue virus envelope proteins. However, glycosaminoglycans are ubiquitously
expressed as cell surface molecules. It is conceivable that HSPG could serve as the
initial docking site for HCV attachment and the virus is subsequently transferred
to another high-affinity receptor (or receptor complex) triggering entry (Barth et
al., 2003).

MECHANISMS OF CELL ENTRY AND FUSION

After attachment, the nucleocapsid of enveloped viruses is released into the cell
cytoplasm as a result of a fusion process between viral and cellular membranes.
Fusion is mediated by specialized viral proteins and takes place either directly at the
plasma membrane or following internalization of the particle into endosomes. The
entry process is controlled by viral surface glycoproteins that trigger the changes
required for mediating fusion. At least two different classes of fusion proteins (I
and II) can be distinguished (Lescar et al., 2001). The flaviviruses enter target cells

18
 Genome and Life Cycle

by receptor-mediated endocytosis and use class II fusion proteins (Lindenbach

and Rice, 2001). By analogy, HCV envelope glycoproteins are believed to belong
to class II fusion proteins (Yagnik et al., 2000). However, in contrast with other
class II fusion proteins, HCV envelope glycoproteins do not appear to require
cellular protease cleavage during their transport through the secretory pathway (Op
De Beeck et al., 2004). HCV entry into cells is pH-dependent and endocytosis-
dependent (Agnello et al., 1999; Bartosch et al., 2003b; Hsu et al., 2003), but the
identity of the HCV fusion peptide remains controversial. E1 appeared as a good
candidate because sequence analysis suggested the presence of a fusion peptide in
its ectodomain (Flint and McKeating, 2000; Rosa et al., 1996). Nevertheless, E2
was shown to share structural homology with class II fusion proteins (Lescar et al.,
2001; Yagnik et al., 2000). Crystallographic 3D structure determination and cryo-
EM-based studies of both envelope glycoproteins are needed to better understand
the mechanisms of HCV fusion.

RNA TRANSLATION AND POST-TRANSLATIONAL PROCESSING

POLYPROTEIN SYNTHESIS
Decapsidation of viral nucleocapsids liberates free positive-strand genomic RNAs
into the cell cytoplasm, where they serve, together with newly synthesized RNAs,
as messenger RNAs for synthesis of the HCV polyprotein. HCV genome translation
is under the control of the IRES, spanning domains II to IV of the 5'UTR and the
first nucleotides of the core-coding region. IRES domain I is not part of the IRES
but plays an important role by modulating IRES-dependent translation (Friebe et
al., 2001; Luo et al., 2003). The IRES mediates cap-independent internal initiation
of HCV polyprotein translation by recruiting both cellular proteins, including
eukaryotic initiation factors (eIF) 2 and 3 and viral proteins (Ji et al., 2004; Lukavsky
et al., 2000; Otto and Puglisi, 2004). Three distinct translation initiation complexes
(40S, 48S and 80S) are generated, as shown by in vitro translation experiments
in HeLa S10 cells and rabbit reticulocyte lysates and by ex vivo experiments in
mammalian cells (Kong and Sarnow, 2002).

The HCV IRES has the capacity to form a stable pre-initiation complex by directly
binding the 40S ribosomal subunit without the need of canonical translation
initiation factors (Otto et al., 2002; Spahn et al., 2001). The 40S subunit assembles
with eIF3 and this ternary complex joins with eIF2, GTP, and the initiator tRNA to
form a 48S particle in which the tRNA is positioned in the P site of the 40S subunit,
base-paired to the start codon of the mRNA. Upon hydrolysis of GTP, eIF2 releases
the initiator tRNA and dissociates from the complex. A second GTP hydrolysis
step involving initiation factor eIF5B then enables the 60S ribosomal subunit to
associate, forming a functional 80S ribosome that initiates viral protein synthesis
(Ji et al., 2004; Kieft et al., 2001; Otto and Puglisi, 2004; Sizova et al., 1998).

19
Chevaliez and Pawlotsky

ER

Fig. 3. Hypothetical HCV replication cycle. HCV particles bind to the host cells via a specific
interaction between the HCV envelope glycoproteins and a yet unknown cellular receptor. Bound
particles are probably internalized by receptor-mediated endocytosis. After the viral genome is liberated
from the nucleocapsid (uncoating) and translated at the rough ER, NS4B (perhaps in conjunction
with other viral or cellular factors) induces the formation of membranous vesicles (referred to as
the membranous web; EM in the lower right). These membranes are supposed to serve as scaffolds
for the viral replication complex. After genome amplification and HCV protein expression, progeny
virions are assembled. The site of virus particle formation has not yet been identified. It may take

20
 Genome and Life Cycle

A number of cellular proteins were reported to interact with the 5'UTR including the
polypyrimidine tract-binding protein (PTB) (Ali and Siddiqui, 1995), heterogeneous
nuclear ribonucleoprotein L (hnRNP L) (Hahm et al., 1998), La autoantigen (Ali and
Siddiqui, 1997), the poly(rC)-binding protein 2 (PCP2) (Spangberg and Schwartz,
1999) and NS1-associated protein 1 (NSAP1) (Kim et al., 2004). The biological
significance of these protein-RNA interactions remains unknown. In addition, HCV
proteins may affect IRES translational efficiency, including the core protein (Zhang
et al., 2002) and non-structural proteins NS4A and NS5B (Kato et al., 2002). The
HCV 3'UTR may also modulate IRES-dependent translation, but this remains
controversial (Imbert et al., 2003; Wang et al., 2005b).

POST-TRANSLATIONAL PROCESSING
HCV genome translation generates a large precursor polyprotein, which is targeted
to the ER membrane for translocation of the E1 ectodomain into the ER lumen,
a process mediated by the internal signal sequence located between the core and
E1 sequences. Cleavage of the signal sequence by the host signal peptidase yields
the immature form of the core protein (P23) (McLauchlan et al., 2002). The signal
peptide is further processed by a host signal peptide peptidase (SPP, a presenilin-
type aspartic protease that resides in the ER membrane) to yield the mature form
of the core protein (P21) (Fig. 3) (Penin et al., 2004b). The host signal peptidase
also ensures cleavage at the E1-E2 junction in the ER lumen. Additional signal
peptidase cleavages at the C-terminal end of E2 and between p7 and NS2 give rise
to p7 (Fig. 3). An incomplete cleavage may lead to the production of non-cleaved
E2-p7 proteins, the role of which is unknown. E1 and E2 subsequently undergo
several maturation steps, including N-glycosylation, conformation and assembly
of E1E2 heterodimers (Penin et al., 2004b). Heterogeneous E1E2 aggregates are
also produced, but their role in viral particle formation is not known.

The zinc-dependent NS2-3 auto-protease ensures cis-cleavage of NS3 from NS2

(Fig. 2). NS3 needs to assemble with its cofactor NS4A to catalyze cis-cleavage at
the NS3-NS4A junction and trans-cleavage at all downstream junctions including
NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B (Fig. 2) (Bartenschlager and
Lohmann, 2000; Lindenbach and Rice, 2005). The cleavage sites recognized by the

place at intracellular membranes derived from the ER or the Golgi compartment. Newly produced
virus particles may leave the host cell by the constitutive secretory pathway. The upper right panel
of the figure shows a schematic representation of an HCV particle. The middle panel shows a model
for the synthesis of negative-stranded (-) and positive stranded (+) progeny RNA via a double-
stranded replicative form (RF) and a replicative intermediate (RI). The bottom panel shows an
electron micrograph of a membranous web (arrow heads) in Huh7 cell containing subgenomic HCV
replicons. The web is composed of small vesicles embedded in a membrane matrix. Bar: 500 nm;
N: nucleus; ER: endoplasmic reticulum; M: mitochondria. Reproduced from Bartenschlager et al.,
2004 with permission.

21
Chevaliez and Pawlotsky

NS3-NS4A protease have in common the following sequence: Asp/GluXXXXCys/

Thr-Ser/Ala, with trans cleavages occurring downstream of a cysteine residue and
the cis cleavage occurring downstream of a threonine residue.

HCV REPLICATION

THE HCV REPLICATION COMPLEX

Infection with a positive-strand RNA virus leads to rearrangements of intracellular
membranes, a prerequisite to the formation of a replication complex that associates
viral proteins, cellular components and nascent RNA strands. The HCV NS4B
protein seems to be sufficient to induce the formation of a membranous web or
membrane-associated foci (Egger et al., 2002; Gretton et al., 2005). It is not known
whether NS4B recruits cellular proteins responsible for vesicle formation or induces
vesicle formation by itself. The membranous web is derived from ER membranes
(Bartenschlager et al., 2004). It is rich in cholesterol and fatty acids, the degree of
saturation of which (that influences membrane fluidity) modulates HCV replication
(Kapadia and Chisari, 2005). HCV replication was shown to occur in detergent-
resistant membranes that co-localize with caveolin-2, an essential component of
lipid raft domains (Shi et al., 2003). Indeed, lipid rafts are involved in the formation
of the replication complex, through protein-protein interactions between hVAP-33
and both NS5A and NS5B HCV proteins (Gao et al., 2004; Shi et al., 2003; Tu et
al., 1999). Overall, the membranous web consists of small vesicles embedded in
a membranous matrix, forming a membrane-associated multiprotein complex that
contains all of the nonstructural HCV proteins (Egger et al., 2002).

MECHANISM OF HCV REPLICATION

The precise mechanisms of HCV replication are still poorly understood. By
analogy with other positive-strand RNA viruses, HCV replication is thought to be
semi-conservative and asymmetric with two steps, both of which are catalyzed by
the NS5B RdRp. The positive-strand genome RNA serves as a template for the
synthesis of a negative-strand intermediate of replication during the first step. In
the second step, negative-strand RNA serves as a template to produce numerous
strands of positive polarity that will subsequently be used for polyprotein translation,
synthesis of new intermediates of replication or packaging into new virus particles
(Bartenschlager et al., 2004). The positive-strand RNA progeny is transcribed in
a five to ten fold in excess compared to negative-strand RNA. NS5B RpRd was
initially thought to catalyze primer-dependent initiation of RNA synthesis, either
through elongation of a primer hybridized to the RNA template or through a copy-
back mechanism (Behrens et al., 1996). More recently, the HCV RdRp was shown
to be capable of initiating de novo RNA synthesis under certain experimental
conditions (Zhong et al., 2000).

22
 Genome and Life Cycle

Initiation of RNA strand synthesis at the 3'-end of the plus and minus strands
involves domain I of the 5'UTR, which can form a G/C-rich stem-loop, the 3'UTR
and a cis-acting replication element (5BSL3.2) consisting of 50 bases located in a
large predicted cruciform structure at the 3' end of the HCV NS5B-coding region
(You et al., 2004). Initiation of RNA replication is triggered by an interaction
between proteins of the replication complex, the 3'X region of the 3'UTR, and
5BSL3.2 that forms a pseudoknot structure with a stem-loop in the 3'UTR (Astier-
Gin et al., 2005; Friebe et al., 2005; You et al., 2004). A phosphorylated form of
PTB was found in the replication complex and PTB was shown to interact with two
conserved stem-loop structures of the 3'UTR, an interaction thought to modulate
RNA replication (Chang and Luo, 2005; Luo, 1999; Luo, 2004). Importantly,
inhibition of PTB expression by means of small interfering RNAs reduced the
amount of HCV proteins and RNA in HCV replicon-harboring Huh7 cells (Chang
and Luo, 2005).

VIRUS ASSEMBLY AND RELEASE

Little is known about HCV assembly and release due to the lack of appropriate study
models. Different variants of the HCV core protein, which can exist as dimeric,
and probably multimeric forms as well, have been shown to be capable of self
assembly in yeast in the absence of viral RNA, generating virus-like particles with
an average diameter of 35 nm (Acosta-Rivero et al., 2004a; Acosta-Rivero et al.,
2004b). Recent reports suggested that the N-terminal portion of the core protein is
sufficient for capsid assembly, in particular the two clusters of basic residues (Klein
et al., 2005; Klein et al., 2004; Kunkel et al., 2001; Lorenzo et al., 2001; Majeau
et al., 2004). In bacterial systems, HCV core proteins efficiently self-assembled to
yield nucleocapsid-like particles with a spherical morphology and a diameter of 60
nm, but the presence of a nucleic acid was required (Kunkel et al., 2001). Overall,
particle formation is probably initiated by the interaction of the core protein with
genomic RNA; HCV core can indeed bind positive-strand RNA in vitro through
stem-loop domains I and III and nt 23-41 (Shimoike et al., 1999; Tanaka et al.,
2000). It is tempting to speculate that the core-RNA interaction may play a role in
the switch from replication to packaging.

Virus-like particles were produced in mammalian cells by using a chimeric virus

replicon allowing high-level expression of HCV structural proteins in BHK-21 cell
lines (Blanchard et al., 2002). Budding of virus-like particles of 50 nm in diameter
in the dilated ER lumen was observed (Blanchard et al., 2003). Transfection of
full-length HCV RNA in HeLa G and HepG2 cell lines led to the formation of
virus-like particles with a diameter of 45 to 60 nm, which were synthesized and
assembled in the cytoplasm and budded into the ER cisternae to form coated particles
(Dash et al., 1997; Mizuno et al., 1995). Indeed, the HCV envelope glycoproteins
E1 and E2 associate with ER membranes through their transmembrane domains

23
Chevaliez and Pawlotsky

(Cocquerel et al., 1998), suggesting that virus assembly occurs in the ER. Structural
proteins have been detected both in the ER and the Golgi apparatus, suggesting that
both compartments are involved in later maturation steps (Serafino et al., 2003).
Moreover, the presence of N-glycan residues at the surface of HCV particles is
also in keeping with a transit via the Golgi apparatus. The mechanisms underlying
exportation of mature virions in the pericellular space have yet to be understood.
Newly produced virus particles may leave the host cell by the constitutive secretory
pathway.

STRUCTURE OF HCV VIRIONS

HCV is thought to adopt a classical icosahedral scaffold in which glycoproteins E1
and E2 are anchored to the host cell-derived double-layer lipid envelope. Within
the envelope is the nucleocapsid which is likely composed of multiple copies of
the core protein, forming an internal icosahedral viral coat that encapsidates the
viral genomic positive-strand RNA. EM and immuno-EM (IEM) studies of bona
fide HCV particles have been hampered by the low amount of viruses in blood and
tissues, the failure to efficiently propagate HCV in cell culture, the poor sensitivity
of these methods, and antibody cross-reactivity. Visualization of HCV virions or
virus-like particles was therefore made essentially from in vitro or non-human in
vivo models.

Infection of primary cells or stable cell lines of hepatic or lymphoid origin with sera
from HCV-infected patients revealed the presence of spherical virus-like particles
(Lacovacci et al., 1997; Serafino et al., 1997; Shimizu et al., 1996). Transfection
of Huh7 cells with full-length HCV genomes did not lead to virion production
(Pietschmann et al., 2002), but virus-like particles were generated after transfection
of HepG2 or Hela G cells (Dash et al., 1997; Mizuno et al., 1995). HCV virus-like
particles could also be produced in mammalian cells, by means of recombinant
Semliki Forest virus (SFV) or vesicular stomatitis virus (VSV) replicons expressing
genes encoding the structural HCV proteins (Blanchard et al., 2003; Ezelle et al.,
2002), and in insect cells infected with a recombinant baculovirus expressing HCV
structural proteins (Baumert et al., 1998; Luckow and Summers, 1988; Maillard
et al., 2001).

MORPHOLOGY OF HCV PARTICLES

Early filtration studies performed in sera from chimpanzees with non-A, non-B
hepatitis suggested that the diameter of the causal agent was in the order of 30-60
nm (He et al., 1987; Yuasa et al., 1991). EM and IEM analysis of particles recovered
from the blood and liver of infected chimpanzees and patients revealed the presence
of spherical particles of 33-70 nm (Bosman et al., 1998; Ishida et al., 2001; Jacob et
al., 1990; Kaito et al., 1994; Li et al., 1995; Petit et al., 2003). Detergent treatment
of infectious sera yielded 30-40 nm icosahedron-shaped particles containing both

24
 Genome and Life Cycle

the HCV core protein and HCV RNA (Takahashi et al., 1992). Virus-like particles
of 45-60 nm was observed in the supernatant of primary cells or stable cell cultures
treated with infectious sera and of cell lines transfected with the full-length HCV
ORF (Dash et al., 1997; Iacovacci et al., 1997; Mizuno et al., 1995; Serafino et al.,
1997; Shimizu et al., 1996). HCV-like particles of 20-60 nm in diameter were also
produced by the expression of HCV structural proteins in cell-free systems (Klein
et al., 2004), SFV replicons (Blanchard et al., 2002; Blanchard et al., 2003), VSV
vectors in rodent BHK-21 cells (Ezelle et al., 2002), bacterial systems (Kunkel et
al., 2001; Lorenzo et al., 2001), baculovirus vectors in insect cells (Baumert et al.,
1998; Xiang et al., 2002) and yeast expression vectors (Acosta-Rivero et al., 2001;
Acosta-Rivero et al., 2004b; Falcon et al., 1999).

The recently developed cell culture system is capable of producing large amounts
of infectious HCV virions (Lindenbach et al., 2005b; Wakita et al., 2005; Zhong
et al., 2005). Two types of viral particles could be visualized in IEM: particles of
30-35 nm in diameter likely to correspond to the viral nucleocapsids, and particles
of 50-60 nm in diameter likely to be the infectious virions (Fig. 4) (Wakita et al.,
2005).

Fig. 4. HCV viral particle produced in a tissue culture system from a cloned viral genome (Wakita et
al., 2005). Viral particles were generated after transfection of the human hepatoma cell line Huh7 by
HCV replicons of the JFH1 genotype 2a strain cloned from a Japanese patient with fulminant hepatitis
(see Chapter 16). HCV particles had a density of 1.15-1.17 g/ml and a spherical morphology with
an average diameter of approximately 55 nm. They were infectious for chimpanzees (Wakita et al.,
2005). The photograph is a courtesy of Ralf Bartenschlager.

25
Chevaliez and Pawlotsky

CIRCULATING FORMS OF HCV VIRIONS

PLASMA COMPARTMENTALIZATION OF HCV PARTICLES

HCV was initially reported to have a lower buoyant density than other members
of the Flaviviridae family on 20-60% isopycnic sucrose density gradients (1.05 to
1.07 g/ml vs 1.15 to 1.25 g/ml, respectively) (Lindenbach and Rice, 2001; Trestard
et al., 1998; Yoshikura et al., 1996). Ultracentrifugation of sera from patients with
acute and chronic HCV infection revealed the presence of two populations of HCV
particles with a broad range of densities, from 1.06 to 1.25 g/ml. Low-density
HCV particles were shown to be principally associated with lipids and lipoproteins
and to contain the infectious virus, whereas high-density HCV particles were
largely associated with immunoglobulins in the form of immune complexes and
supposedly less infectious (Aiyama et al., 1996; Andre et al., 2002; Dienstag et
al., 1979; Hijikata et al., 1993; Thomssen et al., 1992). Interestingly, the respective
proportions of high- and low-density fractions in infected patients' blood were
reported to fluctuate over the course of infection and according to the stage of liver
disease (Choo et al., 1995; Hijikata et al., 1993; Kanto et al., 1994; Kanto et al.,
1995; Petit et al., 2003).

NON-ENVELOPED NUCLEOCAPSIDS
The existence of non-enveloped HCV nucleocapsids during natural infection and
their role in the pathophysiology of HCV infection has been debated. Lipo-viro-
particles (LVPs) rich in HCV RNA, HCV core protein, triglycerides and apoproteins
(especially apoB and apoE) were recently described as large spherical particles of
100 nm, the delipidation of which yielded capsid-like structures (Andre et al., 2002).
Non-enveloped nucleocapsids were detected in the serum of infected patients and in
hepatocytes from patients and experimentally infected chimpanzees (Falcon et al.,
2003a; Falcon et al., 2003b; Maillard et al., 2001). Non-enveloped HCV particles
recovered from the plasma of infected individuals had a buoyant density of 1.27 to
1.34 g/ml (Maillard et al., 2001). They were heterogeneous in size, with a diameter
of 38-62 nm in EM, and were recently shown to exhibit Fcγ receptor-like activity
and bind non-immune IgG (Maillard et al., 2001; Maillard et al., 2004). Whether
or not non-enveloped nucleocapsids are infectious remains to be established.

CONCLUSION
The development of novel anti-HCV therapeutic agents has been stymied by the lack
of an efficient in vitro viral infection system and a suitable animal model. Although
significant progress has been made through genetic and biochemical approaches in
dissecting the molecular processes of HCV replication, our understanding of the
viral entry and virion production steps remains rudimentary. Furthermore, HCV
exists as "quasispecies" in patients due to its high mutation rate and thus viral
resistance will likely be a problem for the emerging small-molecule HCV inhibitors
(Pawlotsky, 2003; Pawlotsky, 2006). The recent development of a robust cell culture

26
 Genome and Life Cycle

system for HCV infection may unravel new aspects of HCV replication, which in
turn will facilitate the development of specific antivirals that target each stage in
the virus life cycle.

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Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland,
S. F., Uprichard, S. L., Wakita, T., and Chisari, F. V. (2005). Robust hepatitis C
virus infection in vitro. Proc Natl Acad Sci USA 102, 9294-9299.
Zhong, W., Uss, A. S., Ferrari, E., Lau, J. Y., and Hong, Z. (2000). De novo initiation
of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J
Virol 74, 2017-2022.
Zibert, A., Kraas, W., Meisel, H., Jung, G., and Roggendorf, M. (1997). Epitope
mapping of antibodies directed against hypervariable region 1 in acute self-
limiting and chronic infections due to hepatitis C virus. J Virol 71, 4123-4127.

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 HCV 5' and 3'UTR

Chapter 2

HCV 5' and 3'UTR: When Translation

Meets Replication
Stephanie T. Shi and Michael M. C. Lai

ABSTRACT
Similar to other positive-strand RNA viruses, the non-coding regions of HCV
RNA, referred herein as 5' and 3' untranslated regions (5'UTR and 3'UTR), contain
important sequence and structural elements critical for HCV translation and RNA
replication. The 5'UTR harbors an internal ribosome entry site (IRES) that directs
viral protein translation via a cap-independent mechanism. As the initiation sites
for RNA synthesis, both 5'UTR and 3'UTR contain signals that are indispensable
for and regulate viral RNA replication. Additional structural elements involved in
translation or RNA replication are also present in both ends of the protein (core and
NS5B)-coding regions. These RNA elements interact with each other either directly
or through the binding of viral and cellular proteins that are most likely involved in
the regulation of translation and RNA replication processes. Since RNA replication
and translation occur on the same RNA molecule, mechanisms must exist to regulate
and separate these two processes. This chapter details the current understanding of
the roles of the UTRs and other structural components in the viral RNA as well as
their binding proteins in HCV translation and RNA replication and speculate on
the possible mechanisms regulating these two different processes.

INTRODUCTION
HCV is a typical flavivirus containing a single-stranded, positive-sense RNA of 9.7
kb in length (Choo et al., 1991). The viral RNA contains a single large open reading
frame (ORF) flanked by an untranslated region (UTR) at each end, a genomic
organization conserved among members of the Flaviviridae family. One of the most
important features of HCV RNA is its high degree of genetic variability as a result
of mutations that occur during viral replication. However, the mutation rate varies
significantly in the different regions of the HCV genome, of which the 5'UTR and
the extreme end of the 3'UTR have the lowest sequence diversity among various
genotypes and subtypes (Choo et al., 1991; Miller and Purcell, 1990; Muerhoff et
al., 1995). The relatively conserved nature of these regions signifies their functional
importance in the viral life cycle.

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A combination of phylogenetic analysis, computer modeling, and chemical and

enzymatic probing has enabled the identification of structural elements in the 5'
and 3' UTRs of HCV RNA. The viral RNA elements (internal ribosome entry
site, IRES) critically involved in the cap-independent translation of HCV RNA
have been analyzed extensively. In contrast, the study of the mechanism of HCV
RNA replication was more limited due to the lack of efficient cell culture or small
animal models. The generation of consensus cDNA clones that are infectious in
chimpanzees provided the first tools for molecular genetic analysis of HCV RNA
replication (Beard et al., 1999; Choo et al., 1989; Kolykhalov et al., 1997; Yanagi
et al., 1997; Yanagi et al., 1999a; Yanagi et al., 1998). Using this approach, the
regions in the 3'UTR that are required for viral replication have been identified
(Kolykhalov et al., 1997; Yanagi et al., 1999b). More recently, the development
of and advances in the cell-based subgenomic replicon system have identified
additional RNA elements of the UTRs and other cis-acting replication elements
(CREs) that are involved in RNA replication and translation (Friebe et al., 2005;
Friebe et al., 2001; Lee et al., 2004a; You et al., 2004).

A number of viral and cellular proteins have been shown to interact with the essential
structural elements in the non-coding and coding regions of HCV RNA and are
presumably involved in the regulation of the viral translation and/or RNA replication
processes. The precise functional roles of most of these proteins have not been
established. The recent development of cell-free HCV RNA replication systems
(Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) provides an additional tool
for studying the viral and host proteins involved in the translation and replication
of HCV RNA, thus identifying novel targets for the development of more effective
antiviral therapies.

STRUCTURAL AND FUNCTIONAL COMPONENTS OF THE HCV

RNA
The 5'UTR and the extreme end of the 3'UTR are the most conserved regions
of HCV RNA in terms of primary sequence and secondary structures. Together
with the fact that these structured domains are located at the 5' and 3' ends of the
genome, it stands to reason that they play important roles in viral RNA translation
and/or replication.

5'UTR
The 5'UTR of the HCV genome is 341-nt long in most viral isolates. There is more
than 90% sequence identity among different HCV genotypes, with some segments
nearly identical among different strains (Bukh et al., 1992). The secondary and
tertiary structures of this region are also largely conserved (Brown et al., 1992;
Honda et al., 1999a; Honda et al., 1996a). The 5'UTRs of HCV, GBV-B (Muerhoff
et al., 1995), and pestiviruses, such as bovine viral diarrhea virus (BVDV) and

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 HCV 5' and 3'UTR

classical swine fever virus, share extensive homology in primary sequence and
secondary structure (Brown et al., 1992; Han et al., 1991; Honda et al., 1996a;
Simons et al., 1995), signifying GBV-B and pestiviruses as the closest relatives
to HCV (Ohba et al., 1996). A combination of computational, phylogenetic, and
mutational analyses of the HCV 5'UTR has identified four major structural domains
(domains I-IV) (Fig. 1), most of which are also conserved among HCV genotypes,
GBV-B, and pestiviruses (Brown et al., 1992; Honda et al., 1999a; Honda et al.,
1996a; Smith et al., 1995). Common features include a large stem-loop III and a
pseudoknot (psk). The 5'UTR sequences of HCV and GBV-B have two smaller
stem-loops, stem-loop Ia near the extreme 5' end and stem-loop IV containing the
translation initiation codon (Honda et al., 1996a).

Fig. 1. The structures of the 5'UTR (Rijnbrand and Lemon, 2000) and 3'UTR (Ito and Lai, 1997;
Kolykhalov et al., 1996) of HCV RNA (represented by the HCV-H strain). The structural diagram of
the 5'UTR was kindly provided by Drs. René Rijnbrand and Stanley Lemon. psk, pseudoknot. The
start codon (nt 342) and stop codon are indicated by boldface characters. The shaded boxes in 5'- and
3'-UTR and are RNA elements putatively involved in RNA replication. The enclosed RNA sequences
in 5'-UTR are the reported elements required for efficient IRES-dependent translation.

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Shi and Lai

The first 40 nt of the 5'UTR constitutes domain I, which is involved in RNA

replication but not essential for translation; therefore, the function of this region
is distinct from the rest of the 5'UTR, which is critical for translation (Friebe et
al., 2001; Luo et al., 2003). The remaining domains II-IV constitute an IRES (Fig.
1) (Brown et al., 1992), which mediates the cap-independent translation of the
HCV ORF (Tsukiyama-Kohara et al., 1992). Domains II and III are relatively
more complex than domain IV and contain multiple stems and loops (Honda et
al., 1999a; Lemon and Honda, 1997). Several electron microscopy (Beales et al.,
2001; Spahn et al., 2001) and NMR studies (Lukavsky et al., 2000) have provided
detailed structural information on the main domains of the IRES. Domains IIIa–IIIc
and II extend in opposite directions from a small central domain that includes stem
loops and junctions IIIe–IIIf (Spahn et al., 2001). The hairpin loop of the small IIIe
subdomain forms a novel tetraloop fold with three exposed Watson–Crick faces
that may be involved in 40S ribosome binding (Lukavsky et al., 2000). The stem of
subdomain IIId forms a loop E motif similar to those observed in prokaryotic and
eukaryotic ribosomal RNA, and a six-nucleotide hairpin loop containing an S-turn
motif (Klinck et al., 2000; Lukavsky et al., 2000). The sequences of the hairpin
loops of subdomains IIIe and IIId are conserved among all HCV isolates and play
an important role in translation initiation.

The base of domain III forms a highly conserved pseudoknot, which is critical
for IRES activity (Wang et al., 1995). Similar pseudoknots with almost identical
primary sequences also exist in the pestiviral and GBV-B IRES elements (Lemon
and Honda, 1997). The pseudoknot is part of the binding site for the 40S ribosome
subunit (Kolupaeva et al., 2000). Another tertiary structural element in domain II,
identified by RNA-RNA crosslinking, may also be involved in ribosome binding
(Lyons et al., 2001). Domain IV is composed of a small stem-loop (stem-loop IV)
in which the initiator codon AUG is located within the single-stranded loop region
(Honda et al., 1996a). Stem-loop IV is not required for internal entry of ribosomes.
In fact, the stability of this stem-loop structure is negatively correlated with the
translation efficiency of the viral RNA (Honda et al., 1996a).

According to a structure-based classification scheme originally designed for

picornaviral IRES elements (Wimmer et al., 1993), the HCV IRES, together with the
IRES elements of the closely related pestiviruses and GBV-B, is classified into type
3 of four existing types (Lemon and Honda, 1997). The picornaviral and flaviviral
IRES elements are significantly different in a number of aspects, suggesting distinct
mechanisms of translation initiation for these two virus families (Rijnbrand and
Lemon, 2000). The picornaviral IRES elements have been shown to be more efficient
than the HCV IRES in directing translation (Borman et al., 1995). In contrast,
viruses in the genus Flavivirus (e.g. yellow fever virus) have significantly shorter
5' UTRs with a cap structure, m7GpppN1mpN2 (Westaway, 1987).

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 HCV 5' and 3'UTR

3'UTR
The 3'UTR of HCV varies between 200 and 235 nt long, which typically consists
of three distinct regions, in the 5' to 3' direction, a variable region, a poly(U/UC)
stretch, and a highly conserved 98-nt X region (Blight and Rice, 1997; Kolykhalov
et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996; Yamada et al., 1996). The
variable region follows immediately the termination codon of the HCV polyprotein,
and is variable in length (ranging from 27 to 70 nt) and composition among
different genotypes. However, it is highly conserved among viral strains of the
same genotype (Kolykhalov et al., 1996; Yanagi et al., 1997; Yanagi et al., 1998).
Computer analysis has identified two possible stem-loop structures in the variable
region, with the first stem-loop extending into the 3' end of the NS5B-coding
sequence (Han and Houghton, 1992; Kolykhalov et al., 1996). The poly(U/UC)
tract consists of a poly(U) stretch and a C(U)n-repeat region (referred to as the
transitional region) and varies greatly in length and slightly in sequence among
different viral isolates (Tanaka et al., 1996). The transitional regions of genotypes
2a, 3a, and 3b have several conserved A residues, which are not present in genotypes
1b and 2b (Tanaka et al., 1996; Yamada et al., 1996; Yanagi et al., 1999a). The
presence of the polypyrimidine tract within the 3'UTR is unique to HCV and GBV-
B (Simons et al., 1995) among flaviviruses. The length of this region has been
correlated with the replication capability of HCV RNA (Friebe and Bartenschlager,
2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). The
X region forms three stable stem-loop structures that are highly conserved across
all genotypes (Blight and Rice, 1997; Ito and Lai, 1997; Kolykhalov et al., 1996)
(Fig. 1). A recent study of the structure of the X region by chemical and enzyme
probing has confirmed the presence of SL1 and SL3, but proposed that the region
between the two stem-loops folds into two hairpins instead of one and may further
form a hypothetical pseudoknot (Dutkiewicz and Ciesiolka, 2005). On the other
hand, the complementary sequence of the X region in this region forms a 3-stem-
loop structure (Dutkiewicz and Ciesiolka, 2005). There is no poly(A) sequence in
the 3'UTR. Instead, the 3'UTR sequence, particularly the X region, is involved in
the regulation of translation, much in the same way as the poly(A) sequence in the
mRNAs of other RNA viruses. Conceivably, these sequences are involved in the
replication, stabilization and also packaging of viral RNA.

As a result of the stem-loop formation in the X region, the HCV genome is predicted
to end with a double-stranded stem. Examination of the 3'-terminal sequences of
the HCV genome in sera from infected patients revealed that most HCV RNAs
contain identical 3' ends with no extra sequence downstream of the X tail (Tanaka
et al., 1996). However, one particular cDNA clone derived from a patient's serum
did contain 2 additional nt (UU), thus generating a single-stranded tail (Yamada et
al., 1996). The structure of the exact 3'-end will have implications for the initiation
of RNA replication.

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OTHER STRUCTURAL COMPONENTS IN THE PROTEIN-CODING REGION

Bioinformatic analysis has revealed the possible presence of additional secondary
structures in other parts of the HCV genome (Hofacker et al., 1998). These include
possible secondary structures in the core- and NS5B-coding regions (Rijnbrand
et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002). Consistent with the
importance of the predicted secondary structures, it has been shown that synonymous
nucleotide mutations are suppressed in the core- and NS5B-coding regions and that
compensatory mutations are frequently observed within the predicted stems (Ina
et al., 1994; Smith and Simmonds, 1997).

The predicted secondary structures within the core-coding region encompass the
first 14 nts of the core gene, which form part of the IRES stem-loop IV (Lemon
and Honda, 1997). There are two more stem-loops between nt 47 and 167 of the
core-coding sequence (nt 391-511 of the genome), which is conserved among all
six HCV genotypes (Smith and Simmonds, 1997). This region, corresponding
roughly to nt 408-929, has been shown to interact with the 5'UTR, resulting in the
reduction of HCV IRES-mediated translation (Honda et al., 1999b). In the NS5B-
coding region (Hofacker et al., 1998; Rijnbrand et al., 2001; Smith and Simmonds,
1997; Tuplin et al., 2002; You et al., 2004), six potential stem-loop structures have
been predicted based on computer modeling (You et al., 2004). The functional
significance of five of these structures in RNA replication has been implicated from
mutational analysis and RNA structure probing in the context of the subgenomic
replicon. Of particular interest is a cruciform structure (5BSL3) at the 3' terminus
of NS5B, which contains three major stem-loop structures, 5BSL3.1, 5BSL3.2,
and 5BSL3.3 (Fig. 2). Its involvement in RNA replication will be discussed in a
later section.

HCV TRANSLATION
Translation of the polyprotein from the HCV RNA genome is the first macromolecular
synthetic event after the viral RNA is released into the cytoplasm of host cells. It
is carried out by a cap-independent mechanism mediated by the highly structured
HCV IRES. The HCV genomic RNA serves as an mRNA for the translation of a
single polyprotein, which is processed by cellular and viral proteases into at least
10 structural and nonstructural proteins (De Francesco et al., 2000).

STRUCTURAL COMPONENTS REQUIRED FOR TRANSLATION

The first 40 nts of the HCV RNA genome, including the first stem-loop domain
(domain I), are not required for translation (Honda et al., 1996b; Rijnbrand et al.,
1995). Instead, deletion of this domain resulted in a stimulation of translation of a
heterologous reporter RNA (Yoo et al., 1992). However, in the context of the HCV
subgenomic replicon, deletion of this domain reduced protein expression by 3 to
5 fold (Luo et al., 2003). In addition, a dinucleotide sequence at nt 34-35 has been
shown to contribute to the differential translation efficiencies between genotype

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 HCV 5' and 3'UTR

Fig. 2. Cis-acting RNA replication regulatory elements in the NS5B-coding region that interact with
the 3'UTR (represented by the HCV-Con1 strain). (A) The cruciform structure formed at the end of
the NS5B-coding sequence contains 5BSL3.1, 5BSL3.2, and 5BSL3.3, among which 5BSL3.2 is
required for HCV RNA replication (You et al., 2004). (B) Kissing-loop interaction between the loop
sequences of 5BSL3.2 and SL2 of the X region (Friebe et al., 2005).

1a and 1b isolates (Honda et al., 1999b). It is, therefore, possible that domain I is
also involved in the regulation of HCV translation in some fashions. The primary
element of the IRES starts at nt 44, which coincides with the 5' border of domain
II (Honda et al., 1999a; Honda et al., 1996b; Reynolds et al., 1995; Rijnbrand et
al., 1995). However, the precise 3' border of the IRES is controversial.

The stem-loop IV of the 5'UTR is predicted to extend into the coding region to
include the first 10 nts (nt 345-354) of the core-encoding gene. Indeed, several
studies have reported the requirement for a short sequence (up to 30 nt) in the core-
coding region for optimal IRES function (Honda et al., 1996a; Hwang et al., 1998;
Lu and Wimmer, 1996; Reynolds et al., 1996). However, efficient translation has also
been observed with certain reporter genes fused immediately after the start codon,
without the core protein-coding sequences (Tsukiyama-Kohara et al., 1992; Wang
et al., 1993). The differences in the conclusions may have been due to the assay
systems and heterologous reporters employed. It has been found that expression of
the reporter gene secretory alkaline phosphatase, but not that of chloramphenicol
acetyltransferase, depends on the presence of downstream core-coding sequences
(Rijnbrand et al., 2001). Conceivably, the core-coding region may contribute to IRES
function by preventing undesirable base pairing of the IRES with other inhibitory
sequences or by promoting favorable protein binding to the IRES. This core-coding

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Shi and Lai

region contains an adenosine-rich stretch, which has been shown to recruit a cellular
protein that enhances the HCV IRES activity (Kim et al., 2004a; Reynolds et al.,
1995). So far, nt 354 is generally regarded as the consensus 3' boundary of the IRES
(Honda et al., 1999a), but the sequence immediately downstream of the IRES (up
to nt 371) may have a stimulating effect on IRES-directed translation. Interestingly,
the core-coding sequences further downstream (near the C-terminal portion) have
been shown to play a negative-regulatory role in HCV translation (Ito and Lai,
1999; Kim et al., 2003; Wang et al., 2000).

Besides the 5'UTR, the 3'UTR sequences, particularly the X region, may also play
a role in HCV RNA translation. It has been shown that HCV RNA containing
the X region was translated 3- to 5-fold more efficiently than the corresponding
RNAs without this region (Ito et al., 1998). The enhancement of IRES-dependent
translation by 3'UTR may be mediated by polypyrimidine tract-binding protein
(PTB), which binds to both the 5' and 3'UTR (Ali and Siddiqui, 1995; Ito and Lai,
1997; Tsuchihara et al., 1997). Since PTB can interact with itself, it can potentially
mediate circularization of HCV RNA, thereby enhancing translation. The role of
the 3'UTR in translation is reminiscent of the poly(A) tail and the poly(A)-binding
protein in the translation of poly(A)-containing mRNAs (Kahvejian et al., 2001).
However, a different study reported that deletion of the poly(U/UC) tract or the
stem-loop 3 of the X region resulted in an enhancement of translation efficiency;
the increase in translation was not mediated by PTB (Murakami et al., 2001).
Additional studies are required to understand the role of the 3'UTR in IRES-
mediated translation of HCV proteins.

THE HCV TRANSLATION MACHINERY

The HCV IRES is responsible for directing the 40S ribosomal subunit in close
contact with the start codon for translation initiation (Lemon and Honda, 1997; Wang
et al., 1993). Enzymatic and chemical footprinting and domain-deletion experiments
have identified domain II and the basal part of domain III, excluding domain IIIb,
as the binding site for the 40S ribosome subunits (Kieft et al., 2001; Kolupaeva et
al., 2000; Lukavsky et al., 2000; Pestova et al., 1998). Although the HCV IRES
with or without domain II recruits the 40S ribosome subunit with comparable
efficiency (Otto et al., 2002), interaction of domain II with the 40S subunit induces
or stabilizes the conformational changes within the ribosome and facilitates the 3´
end of the coding RNA to thread into the mRNA entry channel (Spahn et al., 2001).
The GGG triplet (nt 266-268) of the hexanucleotide (UUGGGU) apical loop of
stem-loop IIId and the pseudoknot are essential for ribosome binding (Kolupaeva et
al., 2000). Mutagenesis studies have also confirmed that the GGG triplet is essential
for IRES activity both in vitro and in vivo (Jubin et al., 2000).

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 HCV 5' and 3'UTR

The viral 5'UTR forms a binary complex with the 40S ribosomal subunit in the
absence of any canonical or non-canonical initiation factors (Pestova et al., 1998).
A ribosomal protein S5, in particular, is important for the efficient translation
initiation of HCV RNA (Fukushi et al., 1997; Fukushi et al., 2001b; Pestova et al.,
1998). Blocking of the S5 binding to HCV IRES interfered with efficient ribosome
assembly at the translation initiation site (Ray and Das, 2004). These features suggest
that HCV IRES uses the prokaryotic mode for forming the mRNA-40S ribosome
complex (Pestova et al., 1998).

Several basal translation initiation factors have been reported to be involved in

the HCV IRES-mediated translation. The eukaryotic initiation factor-3 (eIF3),
alone or together with the 40S ribosome subunit and the eIF2-GTP-initiator tRNA
complex, can specifically interact with the HCV IRES stem-loop IIIb in the absence
of eIF4A, eIF4B and eIF4F, which are required for ribosomal binding during
cap- or EMCV IRES-dependent translation (Kieft et al., 2001; Kolupaeva et al.,
2000; Pestova et al., 1998; Sizova et al., 1998). eIF3 binding is not necessary for
40S-HCV IRES assembly, but is essential for the joining of 60S subunit to form
the active 80S ribosomal complexes (Pestova et al., 1998). These findings suggest
that HCV employs a modified mechanism of IRES-dependent translation. Rabbit
reticulocyte lysates depleted of certain translation factors, such as eIF4G, cannot
support foot-and-mouth-disease virus IRES-, but still can support HCV IRES-
dependent translation (Stassinopoulos and Belsham, 2001). eIF2Bγ and eIF2γ have
also been identified as cofactors of HCV IRES-mediated translation by a functional
genomics approach (Krüger et al., 2000), although their roles in translation have
not been established. These findings combined indicate that HCV IRES-dependent
translation employs a prokaryotic mode for assembling RNA-ribosome complex
and requires only a minimum set of canonical translation factors.

HCV RNA REPLICATION

By analogy with other members of the Flaviviridae, HCV is presumed to replicate
its genome through the production of a full-length negative-strand RNA. Positive-
strand RNAs are then synthesized from the negative-strand template in five- to
ten-fold molar excess over the negative-strand RNA (Lohmann et al., 1999) to be
used in translation, replication, and packaging into progeny viruses. Since RNA
replication has to initiate from the 3'- end of the RNA template of both strands, the
corresponding 5' and 3' UTR of HCV RNA genome likely contains the sequences
required for the initiation and/or regulation of RNA replication.

STRUCTURAL COMPONENTS REQUIRED FOR RNA REPLICATION

Since the 5'UTR is involved in the initiation of both translation and RNA replication,
any possible effects of this region on translation will impact RNA replication
indirectly and vice versa. Therefore, the direct role of 5'UTR in RNA replication

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Shi and Lai

is difficult to assess. Separation of RNA replication and translation was initially

achieved by inserting the IRES elements of poliovirus or classical swine fever virus
between the serially deleted 5'UTR of HCV and the ORF (Friebe et al., 2001; Kim et
al., 2002b; Reusken et al., 2003). The deletions introduced into the 5'-terminal 40 nt
upstream of the IRES region abolished RNA replication but only moderately affected
translation. The first 125 nt of the HCV genome, which includes domain I and II of
the 5'UTR, was shown to be essential and sufficient for RNA replication (Friebe et
al., 2001; Kim et al., 2002b; Reusken et al., 2003). This region overlaps with the
5'end of the IRES. The replication efficiency of RNA was tremendously increased
by the inclusion of the complete 5'UTR (Friebe et al., 2001). Compared with its
close relative BVDV, the requirements for RNA sequences or structures within
the 5'UTR of HCV appear to be more complex because much longer sequences or
particular structures within the IRES are necessary for efficient RNA replication
(Frolov et al., 1998; Wilhelm Grassmann et al., 2005). However, further studies are
required to show whether the sequences downstream are directly involved in RNA
replication or merely contribute to the preservation of the structural and functional
integrity of the minimal replication signal.

Consistent with a role for the 3' terminal nt of the viral RNA in the initiation of
negative-strand RNA, the 3'UTR sequences have been shown to play an essential
role in HCV RNA replication in vitro (Friebe and Bartenschlager, 2002; Yi
and Lemon, 2003a) and in vivo (Kolykhalov et al., 2000; Yanagi et al., 1999b).
The 3'UTR sequences were first shown to be required for the replication of
HCV RNA when deletion of the 3' terminal sequences destroyed the ability of
otherwise infectious synthetic genome-length HCV RNA to initiate infection in
intrahepatically inoculated chimpanzees (Kolykhalov et al., 2000; Yanagi et al.,
1999b). Using a subgenomic HCV replicon, the 3' terminal RNA signals required
for HCV RNA replication were determined to be approximately 225 nt from the 3'
end of the genome (Yi and Lemon, 2003a). The 3'-most 150 nt of the genome, which
includes the 98-nt X region and the 52 nt of the poly(U/UC) tract, are essential for
replication of HCV RNA, while the remaining upstream region of the 3'UTR plays a
facilitating role (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon,
2003a; Yi and Lemon, 2003b). These results suggest an interesting symmetry in the
5'- and 3'- terminal RNA replication signals since the 5'-most domains I and II of
the 5'UTR are essential for replication, while sequences lying further downstream
within the 5'UTR help to facilitate replication but are not absolutely required (Friebe
et al., 2001; Kim et al., 2002b). The X region interacts with the recombinant HCV
RNA polymerase (Cheng et al., 1999; Oh et al., 2000), although other parts of the
3' end of HCV genome may contain additional NS5B-binding sites (Cheng et al.,
1999). The NS5B-binding domain within the X region has been mapped to stem
II and the single-stranded region connecting stem-loops I and II (Oh et al., 2000).
Truncation of 40 nts or more from the 3' end of the X region abolished its template

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 HCV 5' and 3'UTR

activity in vitro (Oh et al., 1999; Oh et al., 2000). A more extensive mutational
analysis of the 3'-end 46 nt that form the terminal hairpin (stem-loop I) in the HCV
replicon provided strong functional evidence for the existence of the structure and
for an essential role of the structure in the replication of HCV RNA (Yi and Lemon,
2003b). It is interesting that the X region is also necessary for efficient translation
of HCV protein (Ito et al., 1998); thus, the same set of sequences are involved in
both RNA replication and translation.

The poly(U/UC) tract is required for HCV RNA replication (Friebe and
Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon,
2003a). It is possible that this region assists in circularizing the viral genome, which
has been shown to be important for efficient RNA replication of other flaviviruses
(Khromykh et al., 2001). This sequence binds several cellular proteins (e.g. PTB),
which may mediate RNA-RNA interaction (Ito and Lai, 1999) and/or the binding
of the replicase complex to RNA. The length of the poly(U/UC) region may
influence viral replication as HCV RNA with a longer poly(U/UC) region had
a replicative advantage in chimpanzees (Kolykhalov et al., 1997; Yanagi et al.,
1999b) than the one with a shorter poly(U/UC). Similar observation was made in
the subgenomic replicon RNAs (Friebe and Bartenschlager, 2002; Yi and Lemon,
2003a). Conversely, the poly(U/UC)-rich sequence may serve as a modulator of
RNA replication under some conditions, as shown in an in vitro RNA polymerase
reaction, in which HCV RNA polymerase stutters at this region (Oh et al., 1999).

The sequences within the variable region of the 3'UTR are not essential for RNA
replication (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b; Yi and Lemon,
2003a), a finding similar to those of other flaviviruses (Khromykh and Westaway,
1997; Mandl et al., 1998; Men et al., 1996). Interruption of sequence integrity within
this region by insertion of the extraneous sequences in this region did not interfere
with the replication of the HCV RNA or replicons (Friebe and Bartenschlager, 2002;
Yanagi et al., 1999b). Nevertheless, deletions in this region impaired the efficiency
of amplification of subgenomic replicons (Yi and Lemon, 2003a).

Some of the conserved RNA elements identified in the NS5B-coding region may
serve as recognition sites for the HCV replicase complex since partially purified
NS5B specifically binds to the coding sequences of NS5B RNA (Cheng et al.,
1999), but their involvement in RNA replication has not been established until
recently. The NS5B-coding region contains a predicted cruciform structure (5BSL3)
consisting of three stem-loops, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Mutations
disrupting the 5BSL3.2 blocked RNA replication, whereas 5BSL3.1 and 5BSL3.3
were shown not to be required for RNA replication (Friebe et al., 2005; You et
al., 2004). Insertion of 5BSL3.2 alone into the variable region of the 3'UTR was
sufficient to rescue RNA replication of a replicon in which all three 5BSLs in the

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NS5B-coding region were disrupted, indicating that 5BSL3.2 can act as a cis-acting
RNA replication element. This insertion allowed the analysis of individual elements
within 5BSL3.2 in more detail without the complication of introducing amino acid
changes in the NS5B-coding region (Friebe et al., 2005).

5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop,
and an 8-base internal loop; the stem structures, but not their primary sequences,
are required for RNA replication (You et al., 2004). In addition, a kissing-loop
interaction between a 7-nt-long complementary sequence in 5BSL3.2 and SL2 in
the X region has been proven essential for RNA replication (Friebe et al., 2005).
In the upper loop of 5BSL3.2, a CACAGC sequence motif is found to be virtually
invariant among HCV genotypes and is also present in cis-acting RNA sequences of
distantly related flaviviruses, such as Kunjin virus, West Nile virus, or Dengue virus
(Markoff, 2003). Given the high genetic conservation in this particular region of the
genome, it may be speculated that certain ubiquitously expressed and evolutionarily
conserved host cell proteins are involved in the formation of a replication complex
that interacts with the 3' end of the flavivirus genome.

INITIATION OF NEGATIVE- AND POSITIVE-STRAND RNA SYNTHESIS

Recombinant NS5B proteins are capable of primer-independent initiation of
RNA synthesis on a variety of virus-specific and nonspecific RNA templates in
vitro (Ferrari et al., 1999; Lohmann et al., 1998; Oh et al., 1999). However, there
are conflicting descriptions of the precise initiation site of negative-strand RNA
transcription on the HCV-specific templates (Hong et al., 2001; Kim et al., 2002a;
Oh et al., 2000; Shim et al., 2002). Oh et al. reported that the transcription of the
negative-strand RNA was initiated within the loop sequence of the 3'X stem-loop
I, at approximately 21 nt from the 3' end of the RNA (Oh et al., 2000). Kim et al.
reported that transcription initiated further downstream, within the 3' stem sequence
of SL1 (Kim et al., 2002a). Shim et al. has shown that transcription can be initiated
by a recombinant NS5B polymerase in vitro at the 3' end of short oligonucleotide
templates representing the 3' terminus of the positive-strand genomic RNA (Shim
et al., 2002). The terminal U is preferred as the initiation nt (Shim et al., 2002),
which is confirmed by the study of a subgenomic replicon (Yi and Lemon, 2003b).
Hong et al. proposed that a unique β-hairpin within the thumb domain of the
NS5B polymerase positions the terminal sequences of the genome so as to initiate
de novo transcription from the 3' terminal nucleotides (Hong et al., 2001). It was
proposed that the β-hairpin ensures the initiation of de novo RNA synthesis at the
3' terminus by preventing movement of the 3' end of the single-stranded RNA
template into the active site of the enzyme. Conceivably, the presence of other
viral and cellular proteins may affect the selection of the initiation point of RNA
replication in vivo.

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 HCV 5' and 3'UTR

The initiation of the positive-strand RNA synthesis has not been as well studied.
Conceivably, the 3' terminus of the negative-strand RNA is essential for positive-
strand RNA replication. In vitro replication studies using recombinant NS5B
showed that the minimal RNA fragment required for efficient replication of the
negative-strand RNA spans nt –239 to –1 (Oh et al., 1999), which is complementary
to domains I to III of the 5'UTR. Various site-specific mutation studies on the
5'UTR of the HCV replicons have revealed the importance of these regions on
HCV genome replication. However, these studies did not distinguish their effects
on either positive- or negative-strand RNA synthesis (Friebe et al., 2001; Kim et
al., 2002b). The predicted secondary structures of positive- or negative-strand RNA
of 5'UTR are slightly different (Schuster et al., 2002). In in vitro RNA synthesis
using the full-length HCV RNA as the template, the NS5B polymerase is capable
of positive-strand RNA synthesis, continuing from the 3' end of the full-length
negative-strand RNA product, resulting in a dimeric hairpin HCV RNA (Oh et al.,
1999). The significance of such a product is not clear.

THE HCV RNA REPLICATION MACHINERY

HCV RNA replication is believed to occur in the cytoplasm of virus-infected
cells based on the cytoplasmic localization of viral RNA (Gowans, 2000) and
polymerase (Hwang et al., 1997; Selby et al., 1993). RNA is synthesized by a
membrane-associated replication complex that includes the HCV RNA-dependent
RNA polymerase (RdRP) NS5B, most of the other viral NS proteins (NS3, NS4A,
NS4B, and NS5A), and possibly cellular proteins (Asabe et al., 1997; Bartenschlager
et al., 1995; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Among the
viral NS proteins, NS4B protein by itself induces membranous alterations that
morphologically resemble the membranous webs found in replicon cells where
viral RNA replication takes place (Egger et al., 2002; Gosert et al., 2003; Shi et
al., 2003). A variety of biochemical evidence suggests that NS4B anchors the
formation of the RNA replication complex (Gao et al., 2004), which is formed on
the detergent-resistant membrane structures containing cholesterol-rich lipid rafts
(Aizaki et al., 2004; Shi et al., 2003). Interestingly, all the nonstructural proteins,
except NS5A, have to be translated in cis from the ORF of the very RNA molecule
in order for RNA replication to occur (Appel et al., 2005). This finding suggests that
the viral proteins are assembled in an ordered and sequential way into the replication
complex soon after translation. The only trans-acting protein NS5A may enter the
replication complex by binding to a cellular protein, VAP-33 (see below).

From the replicon studies, it appears that the RNA replication requires all the HCV
nonstructural proteins except NS2. The NS3 is directly involved in RNA synthesis
probably through its helicase function. The RNA helicase function is presumed to be
necessary for unwinding the secondary structures of RNA template and to separate

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the positive- and negative-strand HCV RNA during replication. The HCV helicase
lies within the C-terminal half of NS3, which has been shown to possess NTPase,
single-stranded (ss) polynucleotide binding, and duplex-unwinding activities (Kim
et al., 1995; Tai et al., 1996). NS3 alone has only a weak RNA unwinding activity,
which can be significantly enhanced by the presence of NS4A (Pang et al., 2002).
The resolution of the crystal structure of NS3 either alone or complexed with
deoxyuridine octamer has provided additional insights into the mechanism of the
HCV NS3 helicase function (Cho et al., 1998; Kim et al., 1998; Yao et al., 1999).

NS5B is a membrane-associated phosphoprotein (Hwang et al., 1997), which

contains signature motifs, such as the GDD, shared by other viral RdRps (Koonin,
1991). The C-terminal 21 aa of NS5B plays a role in anchoring the protein to the
membrane (Yamashita et al., 1998) but also plays a direct role in RNA synthesis
(Lee et al., 2004b; Vo et al., 2004). NS5B also interacts with a SNARE-like cellular
membrane protein, human vesicle-associated membrane protein (VAMP)-associated
protein of 33 kDa (hVAP-33), which may directly or indirectly target the polymerase
to the RNA replication site (Gao et al., 2004; Tu et al., 1999). Reduction of hVAP-33
expression either by dominant-negative mutants or small interfering RNA (siRNA)
of hVAP-33 blocked the association of NS5B with detergent-resistant membranes
and led to an inhibition of HCV RNA replication (Gao et al., 2004; Zhang et al.,
2004).

Although multiple potential phosphorylation sites exist within the NS5B aa

sequence, no site is conserved among all HCV isolates examined (Altschul et
al., 1997), suggesting that phosphorylation of NS5B may vary among different
isolates. Screening of a phage-display library with HCV NS5B protein as bait has
identified one peptide with amino acid sequences homologous to protein kinase
C-related kinase 2 (PRK2) (Kim et al., 2004b). In vitro analysis has revealed that
PRK2 binds and phosphorylates the N-terminal region of NS5B. Further studies in
the subgenomic replicon system have indicated that phosphorylation of NS5B by
PRK2 is involved in the regulation of HCV RNA replication. It is not clear whether
this phosphorylation has an effect on the NS5B polymerase activity and whether
it is conserved among different isolates.

The crystal structure of NS5B shares significant similarity to those of other

polymerases, but also displays certain striking differences (Ago et al., 1999;
Bressanelli et al., 1999; Lesburg et al., 1999). The domain organization in NS5B
can be subdivided into the fingers, palm and thumb, similar to other polymerases.
However, as other polymerases, such as the poliovirus 3D polymerase, are distinctly
U-shaped, the fingers and the thumb domains of NS5B exhibit extensive contacts
between each other, resulting in a globular-shaped molecule. The encircled active
site is relatively inflexible and can accommodate only a template:primer duplex

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without global conformational changes. The C terminus of NS5B (excluding the

hydrophobic tail) is present in the active site of the protein and has been hypothesized
to play a role in the regulation of RdRp activity and template discrimination (Ago
et al., 1999).

In in vitro RdRP assays, NS5B often uses the 3' end of the template RNA or an
artificial oligonucleotide as a primer. (Al et al., 1998; Behrens et al., 1996; De
Francesco et al., 1996; Ferrari et al., 1999; Lohmann et al., 1997; Yamashita et al.,
1998; Yuan et al., 1997). However, it can also initiate de novo RNA synthesis in
a primer-independent manner (Luo et al., 2000; Oh et al., 1999; Sun et al., 2000;
Zhong et al., 2000). NS5B binds in vitro preferentially to several regions in the
3'-end of HCV RNA, including the 3'-coding region of NS5B, the U/UC-rich
sequence, and part of the X region (in the stem I and II) (Fig. 1) (Cheng et al.,
1999; Oh et al., 2000). Partial deletion of the 3'UTR of HCV RNA abolished the
template activity of the RNA (Cheng et al., 1999; Oh et al., 2000). Thus, it appears
that NS5B recognizes some specific sequence or structural elements at the 3' end of
HCV RNA (Cheng et al., 1999; Oh et al., 2000). Once it binds the stem structure of
the 3'UTR, however, NS5B initiates RNA synthesis only from the single-stranded
RNA region closest to the 3' end of the template (Oh et al., 2000). This conclusion
is supported by another study showing that the RdRp reaction mediated by NS5B
requires a stable secondary structure and a single-stranded sequence with at least
one 3'-end cytidylate in the RNA template (Kao et al., 2000).

Since the 3' end of HCV RNA ends with a near-perfect double-stranded stem
(stem I) (Fig. 1), then how does HCV RNA synthesis initiate in vivo, if the in
vitro mechanism reflects the mechanism of RNA synthesis in vivo? There are
several potential mechanisms whereby the 3' end sequence of the viral RNA is
retained during RNA replication: (1) The 3' end of HCV RNA may be extended
by a terminal transferase so that there is a single-stranded tail at the 3' end to allow
NS5B to initiate from the precise 3'-end. Indeed, an HCV cDNA clone containing
two additional nt (UU) at the 3'-end of HCV RNA has been detected (Yamada et
al., 1996). (2) RNA helicase or unwinding proteins may be present in the HCV
replicative complex to unwind the 3'-end stem structure into the single-stranded
region. (3) RNA synthesis may initiate internally in the single-stranded region
within the 3'UTR; the 3'-end sequence may be recovered during the positive-strand
RNA synthesis since the complementary sequence can be made by fold-back RNA
synthesis. (4) The presence of other viral or cellular proteins may alter the choice
of the initiation site of RNA replication.

HCV RdRp activity has been detected in the crude replication complexes prepared
from lysates of cells carrying HCV replicons. This lysate can synthesize RNA from
the endogenous template, but not exogenously added templates, and requires both

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NS5B and NS3 (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). The whole
complex is localized on the detergent-resistant membrane and contains all the
nonstructural proteins of HCV. The viral RNA is enclosed within the membrane
complex and shielded from outside. All the nonstructural proteins are probably
anchored on the membrane structures by a series of protein-protein interactions
between them and with a cellular protein hVAP-33 (Tu et al., 1999). It has been
shown that most of the HCV NS proteins, including NS3, NS4A, NS4B, NS5A,
and NS5B, can interact with each other either directly or indirectly (Asabe et al.,
1997; Bartenschlager et al., 1995; Gao et al., 2004; Ishido et al., 1998; Lin et al.,
1997; Tu et al., 1999). Interestingly, while NS5B interacts with the N terminus of
hVAP-33, NS5A binds the C terminus of hVAP-33. The importance of NS5A in
HCV replication has been further suggested by the detection of a number of adaptive
mutations clustered in a defined region of NS5A in a subgenomic HCV replicon
(Blight et al., 2000). It is conceivable that this region may mediate the interaction
of NS5A with a cellular protein that inhibits HCV replication. Further evidence
supporting the existence of a replication complex consisting of multiple HCV NS
proteins came from an analysis of the adaptive mutations derived from a subgenomic
HCV replicon (Lohmann et al., 2001). An adaptive mutation in NS5B was found
incompatible with those in NS5A or NS4B when introduced back into the same
replicon. These mutations may affect contact sites between these proteins in the
replication complex, resulting in a dramatic reduction in replication efficiency.

REGULATION OF HCV TRANSLATION AND REPLICATION

The 5' and 3' UTRs are clearly the sites of important events leading to the onset of
translation and replication of HCV RNA. The binding of viral or cellular proteins
to the UTRs may modulate the secondary and/or tertiary structure of the viral
RNA to facilitate its recognition by the translation machinery and/or the replicase
complex. These proteins may recruit additional cellular factors and mediate long-
range cross-talks between the ends of HCV RNA.

REGULATION OF TRANSLATION BY VIRAL AND CELLULAR PROTEINS

The HCV IRES-mediated translation is relatively inefficient as compared to that
of other viruses (Borman et al., 1995). It has been suggested that HCV has a self-
modulating mechanism to maintain a low level of replication and translation that
may promote viral persistence. In this regard, it was speculated that domain IV of
the IRES may be stabilized by interaction with the viral core protein, resulting in
translation inhibition (Honda et al., 1996a). It has indeed been shown that the core
protein binds to several sites within HCV IRES, thereby inhibiting translation (Li
et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). However, other studies
have suggested that the core-coding sequence, rather than the core protein itself,
is responsible for the suppression of IRES-mediated translation, possibly through
long-range RNA-RNA interactions with the 5'UTR (Kim et al., 2003; Wang et al.,

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 HCV 5' and 3'UTR

2000). The sites of RNA-RNA interaction have been mapped to nt 24-38 within
the 5'UTR and nt 428-442 of the core-coding sequence (Kim et al., 2003), which
is part of a stem-loop structure (Wang et al., 2000). The stem-loop IV of the IRES
may be one of the candidates for feedback control, since the stabilization of this
structure can reduce IRES activity and the primary sequence within this stem-
loop is conserved in nearly all HCV strains (Honda et al., 1996a). However, these
conflicting reports may have been due to the different reporter RNA constructs
used in the different studies since the stable RNA structure assumed by some
heterologous sequences fused directly at the initiation codon may be detrimental
to translation directed by IRES (Rijnbrand et al., 2001). Furthermore, a cellular
protein PTB binds to the 3'-end of the core-coding region and negatively regulates
HCV translation (Ito and Lai, 1999). Thus, translation can be regulated by multiple
RNA segments and viral proteins.

Several other HCV proteins, E2 (Taylor et al., 1999) and NS5A (Gale et al., 1997;
He et al., 2003), may have an indirect effect on HCV translation by inhibiting PKR,
but the biological significance of this effect is not clear.

Besides the canonical translation factors, such as the 40S ribosomal subunit and
eIF3, the HCV IRES also recruits noncanonical cellular translation factors, such
as La autoantigen (Ali and Siddiqui, 1997) and PTB (Ali and Siddiqui, 1995),
which may regulate translation (Fig. 3). The La antigen is an RNA-binding protein
belonging to the RNA recognition motif (RRM) superfamily (Gottlieb and Steitz,
1989). It has been implicated in various cellular processes (Ford et al., 2001; Gottlieb
and Steitz, 1989) and the translation initiation of picornaviruses and flaviviruses
(Ray and Das, 2002; Wolin and Cedervall, 2002). The La antigen recognizes the
intact HCV IRES structure and significantly augments the IRES-directed translation
in vitro (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004; Pudi et al., 2003; Pudi
et al., 2004). Inhibition of HCV IRES activity caused by sequestration of La protein
can be rescued by the addition of purified La protein (Das et al., 1998; Izumi et
al., 2004). La protein binds to the GCAC motif near the initiator AUG within
stem-loop IV (Pudi et al., 2003). Mutations in the GCAC, which alter the primary
sequence while retaining the overall secondary structure, affect the binding of La
protein to HCV IRES and significantly inhibit IRES-mediated translation both in
vitro and in vivo (Pudi et al., 2004). It has been suggested that the nucleic acid-
dependent ATPase activity of La may promote the transformation of stem-loop IV
into single-stranded conformation, which is favorable for 40S ribosome binding
and the formation of active initiation complex (Lemon and Honda, 1997; Pudi et
al., 2004). In addition, La protein may enhance the binding of the ribosomal protein
S5 to HCV IRES, which, in turn, facilitates the formation of the IRES-40S complex
(Pudi et al., 2004). A recent study suggests that La antigen may also be involved
in HCV RNA replication (Domitrovich et al., 2005).

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Fig. 3. Cellular proteins that interact with HCV RNA. The 5'UTR interacts with a basal translation
factor (eIF3), noncanonical translation factors (PTB and La), and other cellular proteins that may
regulate translation (hnRNP L and PCBP). The numbers in parentheses represent the nt sequence
in the HCV genome, where the proteins bind. PTB has three distinct binding sites in the 5'UTR,
whereas hnRNP L interacts with a region immediately downstream of the AUG codon. Both La
autoantigen and PCBP recognize the entire 5'UTR. There is a PTB-binding site in the core-coding
region, which plays a negative regulatory role in HCV translation. The 3'UTR is bound by a variety
of proteins, all of which interact with the poly(U/UC) region. PTB also binds the X region. The length
of poly(U/UC) affects the replication efficiency (Friebe and Bartenschlager, 2002; Kolykhalov et al.,
1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). These 5'UTR- and 3'UTR-binding proteins may
affect viral replication (HuR, hnRNP C and GAPDH), translation (PTB), or RNA stability (La). VR,
variable region.

PTB interacts with three distinct pyrimidine-rich sequences within the HCV IRES
(Ali and Siddiqui, 1995) (Fig. 3). The interaction of PTB with domain III of the
IRES has been confirmed by electron microscopy analysis (Beales et al., 2001).
Immunodepletion of PTB results in the loss of IRES-directed translation, which,
however, cannot be restored by the addition of purified PTB, suggesting that
additional factors tightly associated with PTB are also required to enhance IRES
activity (Ali and Siddiqui, 1995). In addition to the IRES, PTB has also been shown
to interact with the 3' X region (Ito and Lai, 1997; Tsuchihara et al., 1997) and to
enhance HCV IRES-mediated translation (Ito et al., 1998). This long-range effect
suggests that the HCV 5' and 3'UTR may interact with each other through PTB
or other viral or cellular proteins. Furthermore, the presence of RNA aptamers
of PTB inhibited HCV IRES translation (Anwar et al., 2000). In contrast, results
obtained in a study of the subgenomic replicon system do not support a significant
role of PTB in HCV replication (Tischendorf et al., 2004). However, PTB has
been found in the detergent-resistant membrane complex in cells harboring the
HCV subgenomic replicon, while it is in the detergent-sensitive membrane in

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 HCV 5' and 3'UTR

the control cells, indicating the recruitment of PTB to the HCV RNA replication
complex; knockdown of PTB inhibited HCV RNA replication (Domitrovich et al.,
2005)(Aizaki and Lai, unpublished).

Besides PTB, also known as heterogeneous nuclear ribonucleoprotein I (hnRNP I),

several other proteins of the hnRNP family have been shown to interact with HCV
IRES. hnRNP L specifically interact with the 3' border of the HCV IRES in the
core-coding sequence; the binding correlates with the translation efficiency from
the IRES (Hahm et al., 1998). The mouse minute virus nonstructural protein NS1-
associated protein 1 (NSAP1) (Harris et al., 1999), a homolog of hnRNP R, also
known as SYNCRIP (Synaptotagmin-binding cytoplasmic RNA-interacting protein)
(Hassfeld et al., 1998), was recently shown to enhance IRES-dependent translation
through the interaction with an adenosine-rich region in the 5'-proximal region of the
core-coding sequence (Kim et al., 2004a; Reynolds et al., 1995). This protein appears
to be involved in RNA replication as well (Choi and Lai, unpublished observation).
Poly(rC)-binding protein (PCBP), which is also known as hnRNP E and involved in
the expression regulation of numerous cellular and viral RNAs (Ostareck-Lederer
et al., 1998), interacts with the HCV 5'UTR (Fukushi et al., 2001a; Spångberg and
Schwartz, 1999). PCBP has been implicated in the regulation of poliovirus IRES
activity by binding to the 5'UTR of the viral genome (Gamarnik and Andino, 1998;
Gamarnik and Andino, 2000). However, the specific interaction of PCBP-2 with
the 5' terminal domain I of HCV RNA has no effect on IRES-mediated translation
(Fukushi et al., 2001a). Consistent with the role of domain I in RNA replication
(Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003), PCBP-2 may be
involved in the replication rather than translation of HCV RNA.

Using a functional genomics approach, the proteasome α-subunit PSMA7 has been
shown to be involved in IRES-mediated translation, but it is unknown whether the
protein acts directly on IRES or indirectly through the regulation of other cellular
proteins (Kruger et al., 2001). In summary, multiple cellular proteins binding to the
5' or 3'UTR can regulate HCV translation; some of them regulate both translation
and RNA replication.

REGULATION OF RNA REPLICATION BY VIRAL AND CELLULAR

PROTEINS
All the viral nonstructural proteins except NS2 are required for replication, but
the modes of their participation are not clear. Adaptive mutations in the HCV
replicons that allowed the replicons to enhance replication efficiencies have been
detected in all of the viral NS proteins, particularly NS3 and NS5A, indicating that
every viral nonstructural protein (except NS2) contributes to RNA replication. The
purified recombinant HCV NS3 protein or its helicase domain alone can interact
efficiently and specifically with the 3'-terminal sequences of both positive- and
negative-strand RNA but not with the corresponding complementary 5'-terminal

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RNA sequences (Banerjee and Dasgupta, 2001). Specific interaction of NS3 with
the 3'-terminal sequences of the positive-strand RNA appears to require the entire
3'UTR. A predicted stem-loop structure present at the 3' terminus (nt 5 to 20 from
the 3' end) of the negative-strand RNA, particularly the three G-C pairs within the
stem, appears to be important for NS3 binding to the negative-strand UTR. This
interaction may anchor RNA-protein complexes to the cytoplasmic membrane
where viral replication complexes are formed.

The poly(U/UC)-rich region of the 3'UTR is a hot spot in the HCV genome for
binding cellular proteins (Fig. 3), two of which are the Drosophila melanogaster
embryonic lethal, abnormal visual system (ELAV)-like RNA-binding protein,
HuR, and hnRNP C (Gontarek et al., 1999; Spångberg et al., 2000). Both HuR and
hnRNP C interact with the 3' ends of both the positive- and negative-strand HCV
RNA. Due to its pyrimidine-rich nature, it is not surprising that the poly(U/UC)-rich
region has been identified to interact with PTB (Gontarek et al., 1999; Luo, 1999).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also interacts with the
poly(U/UC) tract (Petrik et al., 1999), but the functional relevance of this interaction
has yet to be determined. Based on studies of hepatitis A virus (HAV), the binding of
GAPDH to the 5'UTR of HAV may directly influence IRES-dependent translation
and/or replication of viral RNA by destabilizing the folded structure of the stem-
loop IIIa of HAV IRES and competing with PTB for the binding to this structure
(Schultz et al., 1996; Yi et al., 2000). The 3'UTR has also been shown to bind La
autoantigen, which protects the HCV RNA from rapid degradation (Spångberg et
al., 2001). Although the role of these proteins in HCV RNA replication has not be
characterized, a group of host factors that bind to the 3'UTR of the closely related
pestivirus BVDV has been shown to be required for viral RNA replication (Isken et
al., 2003). It is conceivable that these cellular proteins are involved in not only RNA
replication but translation as well, possibly through the 5' and 3' UTR interaction,
causing the circularization of the viral RNA.

In addition to viral and cellular proteins, a liver-specific cellular microRNA, miR-

122, was suggested by a recent study to regulate HCV RNA replication by directly
interacting with the 5'UTR (Jopling et al., 2005). A 7-nt sequence (ACACUCC)
complementary to the seed sequence of miR-122 was found in both the 5' and
3' UTRs and predicted to be potential binding sites for miR-122. Disruption of
sequence complementarity between the 5'UTR, but not the 3'UTR, and miR-122
reduced HCV RNA replication without affecting RNA stability or translation. It is
speculated that miR-122 may aid in RNA folding or RNA sequestration in replication
complexes. Since miR-122 is expressed in Huh7 but not HepG2 cells, it may also
play a role in host-range determination.

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 HCV 5' and 3'UTR

THE EVIDENCE FOR DIRECT OR INDIRECT 5'-3' INTERACTION

Translation of vast majority of eukaryotic mRNAs, which are capped at the 5' end
and polyadenylated at the 3' end, has been shown to adopt a closed-loop mechanism,
in which the mRNAs are circularized via a 5'-3' interaction mediated by the cap-
binding proteins, eIF4F and eIF4G, and the poly(A)-binding protein, PABP. The
eIF4G-PABP interaction has also been shown to be required for poly(A)-mediated
stimulation of picornaviral IRES-dependent translation, indicating that the 5'-3'
crosstalk is mechanistically conserved between classical eukaryotic mRNAs and
picornaviral RNA (Herold and Andino, 2001; Michel et al., 2001; Michel et al.,
2000). Circularization has been shown to be important for efficient RNA replication
of other flaviviruses (Khromykh et al., 2001). Even in the absence of a poly(A) tail
in HCV RNA, the closed-loop model may still be preserved in HCV IRES-mediated
translation by the presence of RNA sequences and proteins that can functionally
replace the poly(A) tail and PABP (Ito and Lai, 1999). Indeed, the X region of the
3'UTR has been shown to bind PTB and enhance translation of HCV RNA (Ito et
al., 1998), suggesting that the functions of the X region may be similar to that of
poly(A) in eukaryotic mRNA translation (Kahvejian et al., 2001). Since PTB also
interacts with the 5'UTR, it may mediate crosstalk between the 5'- and 3'-ends
of HCV RNA. Thus, the mechanism of translation enhancement by PTB may be
similar to that of eIF4G-PABP in the translation of cellular and viral RNAs that
contain a poly(A) tail.

SWITCH BETWEEN TRANSLATION AND RNA REPLICATION

Since RNA replication and translation occur on the same RNA molecules, the
question arises how these two processes are coordinated. For some RNA viruses,
there is evidence of coupling between RNA replication and translation. For example,
poliovirus defective-interfering RNA without a translatable ORF can not replicate;
the nature of the protein product is not critical, but the translatability is essential
(Collis et al., 1992; Hagino-Yamagishi and Nomoto, 1989; Novak and Kirkegaard,
1994). This requirement has been demonstrated for several other viral RNAs, such
as clover yellow mosaic virus RNA (White et al., 1992), Kunjin virus (Khromykh
et al., 2000), and rubella virus (Liang and Gillam, 2001). In coronavirus, the cis-
acting protein appears to confer a replication advantage to the RNA; the longer
the ORF, the more robust the RNA replication is (de Groot et al., 1992; Kim et al.,
1993; Liao and Lai, 1995). The mechanism of the coupling of these two processes
is not yet clear. However, there are also viral RNAs (e.g., vesicular stomatitis virus,
influenza virus, Sindbis virus) whose replication does not depend on translation
of the ORF on the same RNA. In any case, translation and replication must be
separated since translation goes in the 5' to 3' direction, whereas negative-strand
RNA synthesis goes from 3' to 5' on the same positive-strand RNA template. When
the translation machinery meets the replication complex in opposite direction,
there must be a mechanism to prevent confrontation. The situation is akin to the
separation of transcription and replication of cellular DNA.

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Shi and Lai

In HCV, the 5' and 3'UTR sequences are involved in the regulation of both
translation and RNA replication. There is substantial overlap in the UTR regions
required for translation and RNA replication. Nevertheless, the structural and
sequence requirement for these two processes may be different. It is conceivable
that the structural changes involved in translation and RNA replication may be
effected by the viral or cellular proteins binding to these regions. Indeed, several
cellular proteins binding to the 5' and 3'UTR of HCV have been shown to affect
both translation and replication.

In poliovirus, a switch between translation and RNA replication has been proposed
to be controlled by PCBP, which enhances translation by binding to the 5'-terminal
cloverleaf structure of the poliovirus RNA, and the viral 3CD polymerase, which
promotes negative-strand RNA synthesis by binding to the same RNA structure,
possibly by altering the structure of this region (Gamarnik and Andino, 1998;
Gamarnik and Andino, 2000). Interestingly, PCBP-1 and 2 have also been shown
to interact with the HCV 5'UTR, with PCBP-2 binding particularly to stem-loop I,
suggesting a possibly similar role of these proteins in regulating a switch between
HCV RNA replication and translation (Fukushi et al., 2001a; Spångberg and
Schwartz, 1999). In addition, the HCV core protein may also be involved in the
switch by down-regulating IRES-dependent translation as a regulatory mechanism
required for the initiation of RNA replication (Li et al., 2003; Shimoike et al.,
1999; Zhang et al., 2002). Since many of the cellular proteins binding to the 5' and
3'UTR of HCV have been reported to regulate both translation and replication, it is
conceivable that the relative ratios of the different proteins may control the switch
between translation and replication. Furthermore, the HCV RNA elements required
for translation and those for replication partially overlap. So, the key question in
this regard is how the structures of these elements are altered by RNA-RNA or
protein-RNA interactions so that the RNA can be properly directed to be used for
translation or replication.

Alternatively, an entirely different mechanism may operate to regulate translation

and RNA replication of HCV. The RNA replication complex has been shown to
reside in the cholesterol-rich, detergent-resistant membrane complex (Aizaki et
al., 2004; Shi et al., 2003), whereas translation occurs on the detergent-sensitive,
endoplasmic reticulum membrane. Thus, there may be separate machineries in
different subcellular compartments for these two processes. The different viral and
cellular proteins may bind to RNA molecules differentially in these two different
compartments. The key question in this regard is how the RNA is transported from
the replication complex to the site of translation or vice versa so that these two
functions can be separated.

70
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PERSPECTIVES
The 5' and 3'UTR are the most conserved regions of HCV RNA and play key roles in
regulating translation and RNA replication. The knowledge on these two processes
is still rudimentary, but the development of subgenomic and genomic replicons and
the infectious culture systems (Lohmann et al., 1999; Wakita et al., 2005) provides
promises for the unraveling of these two processes in the near future. These two
regions also offer promising targets for developing antiviral agents.

Within the past two years, small molecule inhibitors of the NS3 protease and the
RNA-dependent RNA polymerase have been shown in early clinical studies to
be efficacious in both treatment-naïve patients and patients who failed interferon
therapy. However, the extensive genetic heterogeneity of HCV RNA and the rapid
evolution of quasispecies present a substantial challenge for these inhibitors to
broad-spectrum activity. The high degree of sequence conservation in the 5'UTR
and 3'UTR among different HCV genotypes makes these regions attractive targets
for antiviral therapies, such as antisense oligonucleotides (Soler et al., 2004),
ribozymes (Welch et al., 1996; Welch et al., 1998), and siRNAs (Kronke et al., 2004;
Randall and Rice, 2004). The inhibition of HCV RNA translation or replication
has been observed with these inhibitors that target the 5'UTR alone or together
with the core-coding sequence of HCV (Hanecak et al., 1996; Kronke et al., 2004;
Macejak et al., 2000; McCaffrey et al., 2003; Ohkawa et al., 1997; Sakamoto et
al., 1996). Universal siRNAs targeting similar regions have been generated and
proven to be effective against all known genotypes (Kronke et al., 2004; Yokota et
al., 2003). Encouragingly, early clinical trials have demonstrated efficacy of some
of these inhibitors in HCV-infected patients despite the limitations associated with
RNA-based therapies and the inherent structures of the UTR sequences (Branch,
1998; Crooke and Bennett, 1996; Gomez et al., 2004). The interventions directing
against conserved domains of viral RNAs may provide valuable alternatives to
small molecule inhibitors that target HCV proteins.

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Yuan, Z.H., Kumar, U., Thomas, H.C., Wen, Y.M., and Monjardino, J. (1997).
Expression, purification, and partial characterization of HCV RNA polymerase.
Biochem Biophys Res Commun 232, 231-235.
Zhang, J., Yamada, O., Sakamoto, T., Yoshida, H., Iwai, T., Matsushita, Y.,
Shimamura, H., Araki, H., and Shimotohno, K. (2004). Down-regulation of viral
replication by adenoviral-mediated expression of siRNA against cellular cofactors
for hepatitis C virus. Virology 320, 135-143.
Zhang, J., Yamada, O., Yoshida, H., Iwai, T., and Araki, H. (2002). Autogenous
translational inhibition of core protein: implication for switch from translation
to RNA replication in hepatitis C virus. Virology 293, 141-150.
Zhong, W., Uss, A.S., Ferrari, E., Lau, J.Y., and Hong, Z. (2000). De novo initiation
of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J
Virol 74, 2017-2022.

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Chapter 3

Assemble and Interact: Pleiotropic

Functions of the HCV Core Protein
Stephen J. Polyak, Kevin C. Klein, Ikuo Shoji, Tatsuo Miyamura
and Jaisri R. Lingappa

ABSTRACT
While surrogate capsid assembly model systems are currently the best tools for
studying HCV core assembly, bona fide HCV culture systems are being developed.
The time will soon come when HCV culture systems and small animal models will
be the norm, rather than the exception (see Chapters 12 and 16). It is now clear
that HCV core protein interacts with many cellular proteins and signal transduction
pathways, that HCV quasispecies influence biologic responses, and HCV proteins
such as core can have different effects depending on whether the protein is
encountered inside or outside the cell. The studies discussed herein have enhanced
the understanding of HCV capsid assembly and the role(s) of HCV core and host
cell interactions in the establishment of persistent infection and the pathogenesis
of HCV liver disease. Continued studies of this nature will also provide a basis
for the rational design of vaccines and novel therapeutics against HCV infection
in humans.

INTRODUCTION
As covered elsewhere in this book, HCV infection is a serious global health problem,
which accounts for billions of dollars in medical expenses in the US alone (Kim,
2002). Clinically, acute HCV infection is frequently anicteric and asymptomatic.
The situation is compounded given the natural tendency for acute HCV infection
to progress to chronic infection. Thus, more effective strategies to successfully
cure patients of their infection are urgently needed. This chapter focuses on a key
HCV molecule, the HCV core or nucleocapsid protein.

THE CORE OF THE PROBLEM

The HCV core protein has been reported to have many functions. With respect to
the virus, the main function of the core protein is to form the capsid shell that will
house and protect the HCV genomic RNA while the virus passes from one cell
to another, or from one person to another. However, the HCV core protein also
modulates many different host pathways by interacting with a variety of cellular
factors. In the following sections, we will highlight important new developments
in HCV capsid assembly and HCV core-host interactions.

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THE ROLE OF HCV CORE IN CAPSID ASSEMBLY

WHAT IS A CAPSID?
A viral capsid is the protein shell that encapsidates and protects the viral genome.
Viral capsids can be composed of one or more virus-encoded proteins. In the case
of enveloped viruses, after assembling and encapsidating the genomic RNA, a
viral capsid then facilitates virion formation by interacting with the viral envelope
glycoproteins and budding. The budding process is sometimes, but not always,
mediated by the viral capsid. For example, the capsid proteins of Ebola and HIV
contain domains that regulate budding, while in the case of tick borne encephalitis
(TBE) virus, it is the envelope glycoproteins that mediate budding. These events
(capsid assembly, encapsidation, and budding) are typically referred to as late
events in the viral life cycle. For HCV, as will be discussed below, many details of
the late events in the HCV life cycle are unclear.

In the case of HCV, as is true for all members of the Flaviviridae, the core protein
is the only viral protein present in the capsid. The final nucleocapsid contains
genomic RNA, coated and protected by the capsid. HCV, being an enveloped virus,
has a lipid envelope, containing the viral envelope glycoproteins as well as host
membrane proteins, surrounding the nucleocapsid. The late events of the HCV life
cycle, including capsid and virion assembly, are shown schematically in Fig. 1. In
this section, we will focus on HCV core, its characteristics, what is known about
its assembly into a bona fide HCV capsid, and the blocks to HCV capsid assembly
that exist in mammalian cell culture systems.

PROPERTIES OF HCV CORE

The HCV genomic RNA is approximately 9.6 kilobases in length and encodes a
single, large polyprotein of about 3000 amino acids (aa). The polyprotein is cleaved
by viral and cellular proteases to generate at least 10 viral proteins (Suzuki et al.,
1999). The core protein represents the first protein in the polyprotein, followed
by two glycoproteins, E1 and E2. The immature form of HCV core contains 191
aa. These 191 aa have been separated into three general domains (McLauchlan,
2000). The first domain (domain I), encompassing aa 1 - ~122, is highly basic and
very hydrophilic. This domain is thought to be responsible for binding RNA and
mediating capsid assembly, and has been reported to interact with many cellular
proteins. The second domain encompasses the majority of the C-terminus of HCV
core. In contrast to domain I, domain II is hydrophobic. Thus, domain II mediates
interactions with lipids and membrane proteins and is not present in capsid proteins
of most other viruses in the Flaviviridae. The final domain (III), which is very
hydrophobic and is predicted to form an alpha helix, is at the extreme C-terminus
of the immature core protein, and corresponds to the signal sequence for E1 (aa 175
- 191). This domain is cleaved soon after core is translated and is absent from the

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Fig. 1. Overview of capsid/virion assembly. Genomic RNA is translated by a host ribosome. HCV core
is the first polypeptide encoded in the polyprotein. Just proximal to core is the membrane envelope
glycoprotein E1. The signal sequence (SS) for E1 (distal to core) targets the polyprotein to the ER.
Signal peptidase cleaves the immature form of core from the growing polypeptide. Signal peptide
peptidase then cleaves the E1 SS releasing the mature form of core. Core then multimerizes and
encapsidates HCV RNA at the cytoplasmic face of the ER. Capsids that are formed in the cytoplasm
then interact with E1 and bud into the ER lumen. Enveloped virions are then released, presumably
via the secretory pathway.

mature form of HCV core. Nevertheless, domain III appears to be very important
in terms of HCV core stability, targeting, and function. Two major forms of core
protein, corresponding to 21- and 23-kDa (p21 and p23), are generated in vitro and
in cultured cells (Yasui et al., 1998), corresponding to the mature (signal cleaved)
and immature (signal uncleaved) forms of the protein.

HCV CORE BIOGENESIS

Synthesis of HCV core in the same polyprotein as the HCV envelope proteins
creates an interesting predicament in that core, the capsid protein, needs to be
soluble and cytoplasmic, while the envelope glycoproteins are transmembrane
and anchored into the host membrane. Therefore, like other flaviviruses, HCV
has evolved an internal signal sequence for E1, the first envelope glycoprotein
(referred to as E1 SS or domain III, as described above). The E1SS is encoded
between HCV core and E1. Thus, after core is translated, the nascent polyprotein
is targeted to the ER translocation channel by the E1 SS (Fig. 1). A host enzyme
located in the ER, signal peptidase, cleaves just proximal to the E1 SS, releasing
the immature form of core from the polypeptide (Hijikata et al., 1991; Santolini et
al., 1994). A different endoplasmic reticulum (ER) enzyme, signal peptide peptidase
(SPP), subsequently cleaves just before the E1 SS liberating the mature form of
HCV core at the cytoplasmic face of the ER (McLauchlan, 2000; McLauchlan et
al., 2002). SPP is a presenilin-type aspartic protease that catalyses intramembrane
proteolysis of signal sequences and membrane proteins within the ER (Weihofen
et al., 2002). Precise mutational analyses have shown that intramembrane cleavage
by SPP is abolished when helix-breaking and -bending residues in the C-terminal
signal sequence are replaced by basic residues. Furthermore, the signal sequence
itself and three hydrophobic aa Leu-139, Val-140, and Leu-144 of the core protein

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are required for SPP cleavage, although none of these residues are essential for
cleavage at the core-E1 junction by signal peptidase, or for translocation of E1
into the ER (Okamoto et al., 2004). The exact cleavage site for producing mature
core (p21) is still controversial, since Leu-179 (Hussy et al., 1996; McLauchlan et
al., 2002), Leu-182 (Hussy et al., 1996), Ser-173 (Santolini et al., 1994), and Phe-
177 (Okamoto et al., 2004) have all been reported as potential sites of cleavage.
After being cleaved into the mature form at the ER, core can undergo a number of
possible fates, including assembly into capsids, targeting to other organelles, and
interaction with host proteins resulting in modulation of various cellular processes,
as will be discussed in more detail below.

HCV CAPSID STRUCTURE AND PROPERTIES

The main role for HCV core in the viral life cycle is to form a nucleocapsid to
protect the viral genome. Once cleaved from the polyprotein, the mature core
protein presumably assembles into HCV capsids, most likely at the cytoplasmic
face of the ER (Mizuno, 1995; Blanchard, 2002; Blanchard, 2003). Unfortunately,
no cellular system robustly recapitulates late events in the viral life cycle, although
there may be hope with the recent development of an infectious HCV system (see
Chapter 16). For this reason, mechanistic details of this process are lacking. HCV
replicon systems (see Chapter 11), first developed in 1999, represented a major
breakthrough because they allowed replication of HCV RNA in mammalian cells
(Blight et al., 2000; Lohmann et al., 1999). However, even when HCV core is
synthesized to high levels, late events in the HCV life cycle do not occur in most
replicon systems, as judged by electron microscopy (Pietschmann et al., 2002).
Therefore a number of model systems have been developed to study the structure
of HCV capsids and HCV capsid assembly.

Knowledge of HCV capsid appearance in vivo has come from examining particles
in serum or in infected liver biopsies. Non-enveloped capsids have been observed in
the cytoplasm of liver cells, while enveloped particles have been seen in the cisternae
of the ER, as judged by transmission electron microscopy (TEM) (Bosman et al.,
1998; Shimizu et al., 1996). The presence of capsids at or in the ER by TEM in
numerous studies implicates the ER as the site of HCV capsid assembly (Blanchard,
2002; Maillard, 2001; Mizuno, 1995; Shimizu, 1996). More recently, a careful
TEM analysis of HCV virions and non-enveloped nucleocapsids from serum of
HCV infected patients was performed (Maillard et al., 2001). This study revealed
that non-enveloped HCV nucleocapids can be found in significant quantities in
serum. These capsids, as well as those obtained by detergent treatment of enveloped
virions, are spherical but heterogeneous in size, with a bimodal distribution of capsid
diameters corresponding to ~38 - 43 nm and ~54 – 62 nm. It remains unclear what
governs capsid size and whether the size differences are biologically significant.
Unfortunately, unlike with other flaviviruses, visualization of HCV virions or
capsids at atomic resolution has not yet been achieved.

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Biochemical analyses have determined that enveloped HCV virions have a density
1.08 to 1.16 g/ml (Bradley et al., 1991; Kaito et al., 1994; Kanto et al., 1994;
Miyamoto et al., 1992). Similar studies on non-enveloped HCV capsids have
yielded conflicting results. HCV capsids with envelopes removed using detergent
have densities of approximately 1.25 g/ml (Kaito et al., 1994; Kanto et al., 1994;
Miyamoto et al., 1992) or 1.32 - 1.34 g/ml (Maillard et al., 2001; Shindo et al.,
1994), with the electron microscopic appearance of capsids of both densities being
otherwise very similar (Maillard et al., 2001). An explanation has been proposed to
explain the finding of two different buoyant densities: capsids that band at the lower
density (~1.25 g/ml) appear to be associated with fragments of membranes, while
those banding at the higher density (~1.32 g/ml) appear to be free of membranes
(Maillard et al., 2001). However, this hypothesis remains to be tested. Additionally,
it appears that both the immature and mature form of core can assemble and be
incorporated into capsids, although, not surprisingly, the mature form is the main
species in virions (Yasui et al., 1998).

MODEL SYSTEMS FOR CAPSID ASSEMBLY

While electron micrographs of infected serum and hepatocytes give a literal
snapshot of what is occurring in vivo, at the other extreme are minimal systems
that can be used as surrogates for understanding the process of capsid assembly.
In these minimal systems, purified recombinant core is incubated with RNA in
the absence of other cellular factors. In the presence of RNAs containing a high
degree of secondary structure (e.g. tRNA or the HCV 5' untranslated region), C-
terminal truncation mutants were found to assemble into regularly shaped capsids
that resemble HCV capsids from infected individuals (Kunkel et al., 2001). Similar
results were obtained by expressing truncated core constructs in E. coli (Lorenzo
et al., 2001). In contrast, full-length (wild-type) recombinant core assembles into
particles with irregular shapes (Kunkel et al., 2001), raising the possibility that host
factors or co-ordination of assembly with core synthesis may be required to assemble
proper capsids from full-length HCV core. These studies also demonstrated that
domain I is sufficient for core assembly. Furthermore, removal of domain II appeared
to facilitate capsid assembly, allowing the purified core protein to assemble into
more regular-shaped capsids. Together, these systems show that HCV core contains
all of the information to assemble into capsid-like structures (in the presence of
RNA) (Kunkel and Watowich, 2002). However, because of the minimalist nature
of these systems, other systems will be required to determine the mechanism by
which wild-type core assembles into capsids within cells, where assembly is likely
to be influenced by other events including de novo core translation, host factors,
and targeting of core to specific organelles.

A cell-free system, a virtual hybrid between in vitro systems and cellular systems,
has recently been developed to study HCV assembly. In these systems, cellular

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extracts are used to reconstitute and link translation to post-translational events,

such as capsid assembly. Thus, these systems combine the benefits of being able to
manipulate the assembly reaction in a test tube while maintaining a cellular context.
Cell-free systems faithfully reconstituted HCV capsid assembly when full-length
core, either the immature or mature form, was expressed de novo in either wheat
germ extracts or rabbit reticulocyte lysate (Klein et al., 2004). Moreover, TEM
analysis revealed that capsids formed from full-length core in the cell-free system
were morphologically very similar to capsids produced in infected patient serum,
both in size and structure (Klein et al., 2004), thereby validating the cell-free system
for mechanistic and mutational studies. In addition, cell-free HCV capsid assembly
is very efficient, with over 60% of newly-synthesized core polypeptides assembling
into immature capsids (Klein et al., 2004).

Some cellular systems have also been used to study capsid assembly. When over-
expressed in insect cells, core assembles into 30 – 60 nm particles at the ER (Baumert
et al., 1998; Baumert et al., 1999; Maillard et al., 2001) that closely resemble capsids
produced in vivo. When the envelope proteins E1 and E2 are also expressed, capsids
can be seen budding into the ER and cytoplasmic vesicles (Baumert et al., 1998);
however, unfortunately no virus-like particles are released (Baumert et al., 1998;
Baumert et al., 1999; Maillard et al., 2001). Therefore, this system recapitulates
much of what is seen in hepatocytes and supports the notion that capsids assemble
at the ER, although virion production is still blocked at a later step in the viral life
cycle. Nucleocapsid-like particles have also been observed upon expression of
HCV core in yeast (Majeau, 2004).

In contrast to these model systems, in general, mammalian cell lines do not support
HCV capsid assembly. There have been isolated reports of capsids being produced
in cultured mammalian cells (Blanchard, 2002; Ezelle, 20026; Mizuno, 1995);
however, the extent of HCV assembly in these cells is unclear. As noted above,
even in replicon cells with high levels of HCV core synthesis, HCV assembly
is not supported (Pietschmann et al., 2002; Bukh et al., 2002), similar to most
cultured mammalian cells (Hope and McLauchlan, 2000). These findings suggest
that mammalian cell lines either lack a necessary cellular factor(s) or contain
inhibitory factor(s) that cause the majority of core to be targeted away from the
ER, as discussed below. This alternate localization of core (Pietschmann et al.,
2002), possibly in conjunction with other negative regulatory influences, correlates
with failure to assemble HCV capsids or virions in cultured cell lines. Consistent
with this, when crude hepatocyte extracts containing membrane-bound organelles
are added to the highly permissive cell-free capsid assembly system, efficiency of
assembly is reduced (Klein et al., 2004).

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HCV ASSEMBLY: REQUIREMENTS AND MECHANISTIC ANALYSIS

Although an ideal model system for HCV capsid assembly does not exist, much has
been elucidated about the requirements and process of capsid assembly from the
various systems mentioned above. In vitro studies have been useful for structural
analyses, having revealed that HCV core undergoes a conformational change upon
assembling into capsid like structures (Kunkel and Watowich, 2002). Meanwhile, the
cell-free system for HCV capsid assembly has allowed the process of core assembly
to be analyzed mechanistically (Klein et al., 2005; Klein et al., 2004). Pulse chase
analyses in the cell-free system have revealed that assembly occurs very quickly,
with very little delay between completion of translation and completion of assembly
(Klein et al., 2004). Additionally, capsid assembly was not highly dependent on
protein concentration or membranes, unlike many other viruses. When HCV core
expression was decreased 200 fold, only a 2.3 fold decrease in amount of assembly
was observed (Klein et al., 2004). Both the speed of HCV capsid assembly and its
relative concentration independence differ from what has been seen with assembly
of other types of viral capsids (i.e. lentiviruses and hepadnaviruses) in analogous
cell-free systems (Lingappa et al., 1997; Lingappa et al., 1994; Lingappa et al.,
2005). Thus, the basic assembly mechanism of HCV capsids may differ from that
of many other viral capsids that assemble at the cytoplasmic face of membranes.
Assembly may occur in microenvironments, for example on polysomes that contain
a high concentration of core protein translating off a single mRNA. The presence of
high local concentrations of newly-synthesized HCV core polypeptides, possibly in
conjunction with cellular factors, could promote rapid and efficient HCV assembly
in permissive cellular extracts, although future studies will be required to test this
hypothesis.

Model systems for HCV assembly have also been used to define regions of HCV
core that are important for HCV capsid assembly. Studies using recombinant HCV
core truncation mutants have revealed that domains II and III are dispensable for
assembly (Kunkel et al., 2001; Lorenzo et al., 2001). In fact, truncation mutants
lacking these domains assemble better than full-length constructs in vitro (Kunkel
et al., 2001). Systematic analysis of HCV capsid truncation, deletion, and point
mutants in the cell-free HCV capsid assembly system have confirmed that the C-
terminus is dispensable for assembly, and also demonstrated that the N-terminal
68 aa are required for capsid assembly (Klein et al., 2005; Klein et al., 2004).
This region of HCV core contains numerous basic residues organized into two
clusters. Removing either cluster of basic residues, or mutating as few as 4 basic
residues to alanines in either cluster, significantly reduces assembly of capsids in
wheat germ extracts (Klein et al., 2005). Conversely, when neutral aa were deleted
from the same region, no effect on cell-free HCV capsid assembly was observed,
suggesting that the critical determinant for assembly is the overall basic charge of
the N-terminus. Likewise, deletions or mutations in other regions of HCV core

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did not affect assembly (Klein et al., 2005). While these studies indicate that basic
residues in the N-terminus are critical for assembly, it remains unclear whether the
N-terminal 68 residues are sufficient for assembly. It should be noted that other
domains of core are clearly important for interaction of core with cellular factors
and for trafficking of HCV core to distinct cellular locations, as discussed below.
Domains involved in core trafficking and cellular protein interactions are likely to
influence or even regulate HCV capsid assembly in intact cells, but these events
have not been studied together due to lack of cell lines that recapitulate HCV capsid
assembly in a robust manner.

RNA BINDING AND ENCAPSIDATION BY CORE

Besides multimerization to form the capsid, the other major function performed
by core during assembly is RNA encapsidation. Many viruses will encapsidate
non-specific cellular RNA if viral genomic RNA is not present. Moreover, many
viruses use RNA as a scaffold for assembly, and/or to nucleate the assembly process.
HCV core appears to act similarly. Domain I of HCV core is extremely hydrophilic,
largely due to the many basic residues clustered in this region. Basic residues
are frequently involved in nucleic acid binding because the positive charge can
interact with the negative phosphate backbone of nucleic acids. Indeed, HCV core
binds RNA (Fan et al., 1999; Santolini et al., 1994; Shimoike et al., 1999) and this
association is dependent on the basic N-terminus (Santolini et al., 1994). Consistent
with this, and supporting the notion that RNA acts as a scaffold for assembly, RNA
was required for in vitro assembly (Kunkel et al., 2001). Additionally HCV core
has RNA chaperone capabilities, suggesting that core may also help restructure
RNA, which may have implications for specific genomic encapsidation (Cristofari
et al., 2004).

While the notion that HCV core binds to RNA is well established, it is unclear
whether HCV core preferentially binds HCV genomic RNA over cellular RNAs.
Core has been shown to bind ribosomal RNA (Santolini et al., 1994), tRNA (Kunkel
et al., 2001), and HCV genomic RNA (Cristofari et al., 2004; Fan et al., 1999;
Kunkel et al., 2001; Shimoike et al., 1999). It appears that the only requirement
is that the RNA should contain significant amounts of secondary structure. When
recombinant core was incubated with denatured, or unstructured, RNA, it failed
to assemble into capsids suggesting that it could not interact with unstructured
RNA. Conversely, when highly structured tRNA or the HCV UTR was used, core
assembly was promoted (Kunkel et al., 2001).

If core binds to any structured RNA, how does genomic RNA get specifically
packaged? Many viral capsid proteins have a higher affinity for specific structures in
their cognate genomic RNA, allowing them to preferentially bind the proper RNA.
It is unclear whether HCV core has higher affinity for HCV genomic RNA. One

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study demonstrated that the HCV core protein binds specifically to a radiolabeled
probe containing the 5' UTR of the genomic RNA. This interaction was abolished
by excess unlabeled probe, but not by unlabeled, non-specific RNA, suggesting
that core preferentially binds genomic RNA (Fan et al., 1999). This could explain
how genomic RNA gets selectively packaged into virions over other cellular RNAs.
Conversely, Santolini et al. reported that core fusion proteins bind equally well to
HCV genomic RNA and heterologous RNA, suggesting that HCV core does not
have enough specificity in its binding to promote genomic RNA encapsidation
(Santolini et al., 1994). If HCV core does not specifically bind genomic RNA, then
some other mechanism must exist to promote encapsidation of the genome. One
possibility is that assembly occurs in microenvironments that contain only a single
species of mRNA (i.e. HCV genomic RNA), as discussed above. Unfortunately,
RNA encapsidation has not yet been analyzed in conjunction with capsid assembly
in any system, so it remains unclear exactly what RNAs are encapsidated and how
HCV core selects RNA for encapsidation during synthesis and assembly.

CAPSID ASSEMBLY: LIGHT AT THE END OF THE TUNNEL?

As mentioned, for the most part cell culture systems do not support virion production,
or even capsid assembly. However, isolated reports have identified infectious virus
propagated in special cell culture systems and at low levels. One group infected
hepatocytes that were cultured in a radial-flow bioreactor and found that HCV is
able to replicate to very low titers (Aizaki et al., 2003). Additionally, at the 11th
International Meeting on Hepatitis C and Related Viruses in Heidelberg in October
2004, there were three reports of very low titer infectious virus particle formation
in cells transfected with HCV genomic RNA (Murakami et al., 2004b; Pietschmann
et al., 2004; Wakita et al., 2004). These initial studies have been confirmed by
independent groups (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al.,
2005) (See Chapter 16). Use of 3-dimensional cultures (Murakami et al., 2004b)
or transfection with the JFH strain (Pietschmann et al., 2004; Wakita et al., 2004)
resulted in production of infectious particles. In one case infection was receptor
mediated, as antibodies to the putative HCV receptor, CD81, blocked infection
(Pietschmann et al., 2004). Unfortunately, in all cases, levels of virus production
were too low to result in measurable titers or any ultrastructural evidence of virus
formation (Aizaki et al., 2003; Murakami et al., 2004b; Pietschmann et al., 2004;
Wakita et al., 2004). Most recently, Heller et al. also isolated virus like particles from
cell culture after transfecting RNA corresponding to the exact genomic sequence
(Heller et al., 2005). This study also showed morphologic data, which suggests
that the particles produced are, indeed, virions. Thus, a new wave of data shows
evidence that HCV can assemble into capsids and, subsequently, into virions in
mammalian cell culture. However, it is unclear whether assembly in these systems
is just a stochastic event, what amount of virus or virus like particles are produced,
how assembly is regulated in these systems, or what cellular subpopulation, if

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any, is producing the limited number of viruses. By using a combination of all

of the current model systems, as well as, newly described cellular systems, new
insights into the mechanism by which HCV assembly is regulated in cells should
be elucidated. This could allow for enhancements of current cell culture systems
that could in turn facilitate the study of late events of the HCV life cycle in cells.

SUB-CELLULAR TARGETING OF HCV CORE

IF CORE DOES NOT ASSEMBLE, WHERE DOES IT GO?

While much information has been elucidated from the various model systems
outlined above, surprisingly, mammalian cell lines including human liver-derived
cell lines fail to produce quantifiable levels of HCV capsids, or virions. For reasons
that remain unclear, in these cell lines HCV core polypeptides can be directed to
alternate cellular locations upon release from the nascent HCV polyprotein. The
fact that core assembles efficiently in infected humans and chimpanzees, but not in
intact cultured cell-lines, suggests that HCV assembly can be negatively regulated.
Trafficking of core to alternate sites is one possible mechanism for negative
regulation of capsid assembly.

One approach to studying the subcellular localization of core involves immunostaining

liver biopsy specimens from infected patients. This has revealed that the core protein
predominantly localizes within the cytoplasm of infected hepatocytes, and often
shows a punctate granular distribution within cells (Gonzalez-Peralta et al., 1994;
Gowans, 2000; Sansonno et al., 2004; Yap et al., 1994). However, when the core
protein alone or the entire viral polyprotein are expressed in mammalian cells,
the majority of core has been observed at the ER membrane (Lo et al., 1995), on
the surface of lipid droplets (Barba et al., 1997; Hope et al., 2002; McLauchlan
et al., 2002; Pietschmann et al., 2002; Shi et al., 2002), and on mitochondrial and
mitochondrial-associated membranes (Schwer et al., 2004; Suzuki et al., 2005).
In addition, core is also known to target to the nucleus (Lo et al., 1995; Matsuura
et al., 1994; Moriishi et al., 2003; Moriya et al., 1998; Yasui et al., 1998), where it
can be a substrate for proteasomal degradation.

What governs whether core stays at the ER to assemble or traffics to other areas of
the cell is not completely understood. Nevertheless, it is clear that such regulation
exists and is quite complex. The finding that core targets to lipid droplets and
mitochondria, but E1 and E2 do not (Pietschmann et al., 2002; Schwer et al., 2004),
raises the possibility that targeting of core away from the ER occurs at a very early
time after core synthesis, before core has had time to interact with the envelope
glycoproteins. Furthermore, a number of studies suggest that aa in domains II and
III direct the post-translational trafficking of core, although agreement is lacking
as to which residues are critical. Okamoto et al. has shown that not only the C-

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terminal signal sequence but also aa 128-151 are required for ER retention of the
core protein by using a series of N-terminally truncated core protein constructs
(Okamoto et al., 2004). Suzuki et al. has reported that a region of aa 112-152
mediates association of the core protein with the ER in the absence of the C-terminal
signal sequence (Suzuki et al., 2005). McLauchlan et al. have proposed that a large
part of the core protein remains within the cytoplasmic leaflet of the ER membrane
after SPP cleavage (McLauchlan et al., 2002). Upon intramembrane cleavage of
the transmembrane signal peptide, the processed core protein may traffic along the
lipid bilayer from the site of biosynthesis to zones at the ER, where lipid droplets
are produced (McLauchlan et al., 2002).

Deletion analyses have revealed that domain II (in particular residues between aa 125
- 144) plays a critical role in targeting core to lipid droplets (Hope and McLauchlan,
2000; Hope et al., 2002). Notably, no domain homologous to domain II is present
in the core proteins of related pesti- and flavi-viruses. In contrast, the core protein
of GB Virus B, from the GB virus group within the Flaviviridae, does contain a
homologous domain that also appears to mediate targeting to lipid droplets (Hope et
al., 2002). Domain II contains two closely spaced prolines that form a proline knot
and appear to be required for targeting core to lipid droplets. The region containing
this proline knot can be replaced with a proline knot domain from lipid-associated
plant proteins called oleosins (Hope et al., 2002), with preservation of lipid
targeting. Lipid targeting of HCV core can also be altered by mutations that affect
SPP cleavage. Helix-breaking point mutations within the signal sequence (domain
III) eliminate SPP cleavage, but also eliminate trafficking to lipid droplets, leaving
core protein on the cytoplasmic face of the ER (McLauchlan et al., 2002; Okamoto
et al., 2004). While these alternate pathways for core trafficking are beginning to
be defined, the downstream consequences of different post-translational trafficking
pathways on core function have not yet been explored. This is in part because using
core mutants to study these cellular fates has proven to be relatively tricky. Studies
have shown that C-terminally truncated versions of the core protein are localized
exclusively to the nucleus (Suzuki et al., 1995). A fraction of the core protein was
detected in the nucleus even when full-length HCV core gene was expressed,
suggesting that the mature core protein also localizes to the nucleus (Moriya et
al., 1997a; Yasui et al., 1998). The N-terminal domain of the core protein contains
three stretches of arginine- and lysine- rich sequences. These basic-residue stretches
function as nuclear localization signals (NLSs) for translocation of the core protein
to the nucleus (Chang et al., 1994; Suzuki et al., 1995). Each of the NLS motifs
of the core protein is able to bind importin-α. At least two of them are required
for efficient nuclear distribution of the core protein in cells, suggesting that they
constitute a bipartite NLS (Suzuki et al., 2005).

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The major fate of core that is targeted to the nucleus is degradation by the nuclear
proteasome (Hope et al., 2002; McLauchlan et al., 2002; Moriishi et al., 2003).
Whether this is a cellular protein "quality control" mechanism, a normal pathway
for core, or a pathway with other functional consequences is unclear. Nevertheless,
it appears that constructs encoding mutations in the C terminus of core are less
stable in cells than is wild-type core (Moriishi et al., 2003). McLauchlan and
colleagues have proposed that the ability of domain II to mediate attachment
of core to lipid droplets also protects core from degradation. Furthermore, they
demonstrated that core constructs encoding a deletion in domain II are protected
from degradation when they also encode a mutation that blocks cleavage of domain
III by SPP (McLauchlan et al., 2002). Related to this observation, the mature
form of core is much less stable when expressed as such than when expressed as
the immature form of core which transiently contains domain III (E1 SS) before
undergoing processing (Suzuki et al., 1995; Suzuki et al., 1999; Suzuki et al., 2001).
Therefore, while the final product is the same, the presence of domain III during
core biogenesis greatly influences core stability. Domain III, while not present
in the mature wild-type core protein, plays a complex and important role in core
stability. Like domain II, domain III and its cleavage may be involved in linking
HCV core to cellular pathways that target it to other regions of the cell and protect
it from degradation. Interestingly, although truncations and deletions in domain II
lead to rapid degradation in mammalian cells, this phenomenon is not seen in cell-
free capsid assembly systems, even when mammalian cell extracts are used (Klein
et al., 2005; Klein et al., 2004). This is likely due to the absence of the nucleus in
these systems, which prevents targeting to the nuclear proteasome, and allows such
mutants to be expressed and analyzed.

Core appears to be peripherally associated with mitochondria, since it is accessible

to protease digestion and carbonate extraction (Schwer et al., 2004) as is the case
at the ER (McLauchlan et al., 2002). Most likely, core traffics from the ER to both
the mitochondria and lipid droplets via membrane bridges, since both of these
compartments are likely derived from the ER. Mitochondrial targeting appears to be
governed by an aa sequence in core. Schwer et al. demonstrated that a short stretch
extending from aa 149-158 located in domain II governs mitochondrial targeting
(Schwer et al., 2004). Suzuki et al. reported that a region of 41 residues from aa
112-152 is responsible for association between the core protein and mitochondria
(Suzuki et al., 2005). This discrepancy may be due to the differences of HCV clones
and experimental settings.

POST-TRANSLATIONAL MODIFICATIONS OF HCV CORE

Post-translational modification plays crucial roles for regulating the function of the
proteins. Several studies have shown post-translational modification of HCV core
protein. Phosphorylation of the core protein in insects cells (Lanford et al., 1993),

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reticulocyte lysates (Shih et al., 1995), and mammalian cells (Lu and Ou, 2002)
have been reported. Cellular protein kinase A (PKA) and protein kinase C (PKC)
were identified as possible protein kinases responsible for phosphorylation of HCV
core protein. Phosphorylation at Ser-116 may regulate nuclear localization of the
core protein (Lu and Ou, 2002).

Post-translational modification of the core protein by tissue transglutaminase has

been reported (Lu et al., 2001). Tissue transglutaminase catalyzes the formation of a
γ-carboxyl-ε-lysine isopeptide bond by joining the γ-carboxamide group of glutamine
to the amino group of lysine. A small fraction of the core protein has been shown to
form a dimer that is highly stable and resistant to denaturation and reduction by SDS
and β-mercaptoethanol. A potential role for tissue transglutaminase in core dimer
formation has been proposed (Lu et al., 2001). The ubiquitin-proteasome pathway
is the major route by which selective protein degradation occurs in eukaryotic cells
and is now emerging as an essential mechanism of cellular regulation (Finley et
al., 2004; Hershko and Ciechanover, 1998). As mentioned above, the core protein
is targeted for ubiquitination and degradation by an unknown ubiquitin ligase. The
C-terminus of the core protein is important for regulating stability of the protein
(Kato et al., 2003; Suzuki et al., 2001). When the core protein is expressed as the
C-terminal truncated forms such as aa 1-173 (21kDa) and 1-152 (17kDa), the core
protein is unstable (Kato et al., 2003; Moriishi et al., 2003; Suzuki et al., 2001).
Specific proteasome inhibitors stabilize these short-lived forms of the core protein,
suggesting that the proteasome machinery is responsible for their degradation (Fig.
2). By contrast, the full-length form of the core protein (aa 1-191) is long-lived.
Only the C-terminal truncated form of the core protein can be multi-ubiquitinated,
and the predominant stable form of the core protein links to a single or only a
few ubiquitin moieties (Suzuki et al., 2001). To understand the mechanism of
ubiquitination of the core protein, the specific E3 ubiquitin ligase that acts on HCV
core has to be identified.

A proteasome activator, PA28γ, has been identified as a core-binding protein by yeast

two-hybrid screening (Moriishi et al., 2003). PA28γ can interact with the core protein
in cultured cells, as well as in the liver of transgenic mice and chronic hepatitis
C patients. PA28γ predominates in the nucleus and forms a homopolymer, which
associates with the 20S proteasome (Tanahashi et al., 1997), thereby enhancing
proteasome activity (Realini et al., 1997). Over-expression of PA28γ enhanced
proteolysis of the core protein, suggesting that PA28γ affects proteasomal activity
and regulates stability of the core protein (Moriishi et al., 2003) (Fig. 2). Evidence
has been accumulating that ubiquitin-proteasome pathway plays a crucial role in
the viral life cycle and in pathogenesis (Banks et al., 2003; Scheffner et al., 1993).
However, the biological significance of ubiquitin-dependent degradation of the
core protein remains to be elucidated.

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Fig. 2. A model for the processing of HCV precursor and degradation of the core protein by the
Ubiquitin-proteasome pathway. The junction between core and E1 is cleaved by the signal peptidase,
resulting in production of p23 form of the core protein. Additional cleavage of the core protein by
signal peptide peptidase produces p21 form of the core protein. Further processed forms of the core
protein, such as p17, are produced by unknown mechanisms. The C-terminal truncated form of the
core protein is poly- ubiquitinated by an unidentified E3 ubiquitin ligase and targeted for proteasomal
degradation. The immature core protein links to a single or a few ubiquitin moieties and is long-lived.
A proteasome activator, PA28γ, enhances proteasomal degradation of the core protein.

HCV CORE-HOST INTERACTIONS

Core-host interactions will be discussed in terms of their affects on host antiviral
and immune responses, and HCV pathogenesis. The recent finding of core protein
in the serum on infected patients has forced one to think that HCV host interactions
not only occur within infected cells, but they can also occur extracellularly.

EFFECTS ON T CELL FUNCTION

HCV infection in humans is almost invariably associated with viral persistence
leading to chronic hepatitis – predisposing the host to development of cirrhosis

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and hepatocellular carcinoma. CD8+ T cells play a pivotal role in controlling

HCV infection; but, in chronic HCV patients, severe CD4+ and CD8+ T cell
dysfunction has been observed (Shoukry et al., 2004). This suggests that HCV
may employ mechanisms to evade or possibly suppress the host T cell response.
In exploring the possible evasion mechanism(s) in order to design strategies for
therapeutics and improved immunization, the HCV core protein was identified as
an immunomodulatory molecule suppressing T lymphocyte responsiveness through
its interaction with complement receptor (gC1qR) (Kittlesen et al., 2000).

It was demonstrated that the HCV core protein suppresses an in vivo anti-viral CD8+
T cell response to vaccinia virus, and inhibits the production of IFN-γ and IL-2 in
an experimental murine model. A host target protein (gC1qR) on T cells was shown
to bind HCV core. Like the natural ligand, C1q, the binding of extracellular core to
gC1qR displayed on T cell surface lead to CD4+ T cell deregulation and suppression
of CD8+ T cell function. Importantly, HCV core-gC1qR ligation induced the
expression of negative signaling molecules (e.g. SHP-1 and SOCS1) in CD4+ T
cells. The data suggest that core has potent immunomodulatory functions.

EFFECTS ON TOLL-LIKE RECEPTORS

Cells sense the presence of extracellular pathogens via cell surface toll-like receptors
(TLRs). There are approximately 10-15 TLRs in mammals, which are responsible
for sensing microbial infection, via recognition of pathogen associated molecular
patterns (PAMP), such as lipopolysaccharide (LPS; TLR4), double-stranded RNA
(dsRNA; TLR3), CpG DNA of bacteria (TLR9), and single-stranded RNA (ssRNA;
TLR7) (Iwasaki and Medzhitov, 2004). After binding pathogens, TLR signaling
involves coupling of toll-IL-1 receptor (TIR) containing adapter proteins such
as TIRAP, TRIF, TIRAP and MAL, and activation of signaling molecules IL-1
receptor associated kinase (IRAK), MyD88, and TNF receptor-associated factor
6 (TRAF-6). Ultimately, transcription factors such as mitogen activated protein
kinases (MAPK), NF-κB, and IRF-3 become activated, leading to production of
IFN-α/β (Hertzog et al., 2003). Interestingly, DC maturation in vitro is impaired
in chronic HCV infection when compared to those subjects with spontaneously
resolved infection and normal controls (Anthony et al., 2004; Dolganiuc et al.,
2003; Kanto et al., 2004; Murakami et al., 2004a; Sarobe et al., 2003; Tsubouchi et
al., 2004a; Tsubouchi et al., 2004b; Wertheimer et al., 2004). Recent studies have
provided mechanistic insights into these events.

In a study of the effect on the immunostimulatory effects of lipopeptides, 10 of 14

and 9 of 14 HCV core lipopeptides stimulated a reporter gene in TLR2-expressing
and TLR4-expressing cells but not in mock-transfected control cells (Duesberg
et al., 2002). However, activation was dependent on the lipid moiety since the
same free peptides had no stimulatory effect on the TLR2 or TLR4 transfected

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cells. A study by a different group found that addition of recombinant HCV core
protein to human monocytes, and human embryonic kidney cells transfected with
TLR2 triggered inflammatory cell activation and failed to activate macrophages
from TLR2 or MyD88-deficient mice (Dolganiuc et al., 2004). HCV core induced
interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation
of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and
induced AP-1 activation. Cell activation required core aa 2-122. Interestingly,
HCV core protein was also taken up by macrophages, but this was independent of
TLR2 expression. These data indicate that the HCV core protein can trigger innate
immune responses.

EFFECTS OF HCV CORE ON THE INTERFERON SYSTEM

Several studies have documented that the HCV core protein can activate the
interferon (IFN) system. For example, core activates the IFN stimulated genes (ISG)
2-5 OAS (Naganuma et al., 2000) and PKR (Delhem et al., 2001). PKR and 2-5 OAS
are two major ISGs that mediate the IFN antiviral response against many viruses. It
was also recently shown that HCV core protein activates the innate antiviral cellular
response involving interferon regulatory factors (Miller et al., 2004). Core induced
IRF-1 transcription and mRNA expression, and caused dose-dependent induction of
the IFN-β promoter and IFN-β mRNA expression. In the presence of IFN-α, core
expression caused increased IFN-stimulated gene factor 3 (ISGF3) binding to the
IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1.
Core expression also activated IFN-γ signaling (Miller et al., 2004).

The effects of core on innate cellular antiviral responses including TLR and IFN
pathways may be critically important during acute infection. Following binding,
internalization, and uncoating of HCV virions, core, in the form of nucleocapsid,
is the first viral protein to interact with the intercellular milieu of cellular proteins
and signaling pathways. Because core mutates during virus replication, HCV core
is present as a quasispecies in infected patients (Pawlotsky, 2003). What is not clear
at present is whether HCV core's inherent variability influences innate antiviral
responses such as TLR signaling and IRF-Jak-Stat activation. Fig. 3 suggests that
there is indeed heterogeneity in innate antiviral responses to genetically different
HCV core isolates. Fig. 3A depicts the sequence of 2 core proteins (named Core
1 and Core 2) derived from 2 different genotype 1b infected patients. As shown
in the figure, the two isolates differed by 7 aa. The 2 core genes were engineered
into a tetracycline regulated expression vector, such that in the absence of
tetracycline in the medium, both Core 1 and Core 2 proteins were expressed in
HeLa cells. Addition of tetracycline to the medium blocked core expression. Fig.
3C presents the effects of Core 1 and Core 2 expression on transcription of an IFN
responsive promoter, the ISRE. In the absence of IFN, expression of Core 1 was
associated with a 3-fold increase in activation of the ISRE, compared to when

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Fig. 3. Effects of HCV Core Protein Expression on Type I IFN Signal Transduction. A, the sequence
of the Core 1 and Core 2 genes are aligned. B, Tetracycline regulated expression of the Core 1 and
Core 2 proteins in HeLa cells. Plasmids were transfected into HeLa tet-off cells, grown in the absence
and presence of tetracycline to induce and repress core protein expression, respectively, and protein
lysates were subjected to Western blot analysis at 48 hours post-transfection. C, Differential effects
of Core 1 and Core 2 proteins on ISRE activation. pTRE-Core 1 and pTRE-Core 2 plasmids were
cotransfected with an ISRE-luciferase reporter plasmid into HeLa tet-off cells, incubated in the
presence or absence of tetracycline for 40 hours, and treated with or without 500 U/ml of IFN-α for
6 hours. Luciferase activity was determined on equal amounts of protein lysates.

gene expression was repressed. In the presence of IFN, Core 1 induced a 2-fold
increase in luciferase activity. Expression of Core 2 resulted in only marginal ISRE
stimulation. These data demonstrate that 2 genetically different HCV core proteins
activate a canonical IFN promoter to varying degrees. The data suggest that HCV
quasispecies differentially modulate host cell responses. Indeed, other studies have
demonstrated that NS5A mediated transcriptional activation varies among clinical
quasispecies isolates (Pellerin et al., 2004). Thus, future studies should take into
account genetic and structural heterogeneity of HCV isolates as being important
factors in host responsiveness to HCV infection.

This concept may have clinical implications. It can be hypothesized that genetic
and structural variants of HCV proteins such core could differentially trigger

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innate antiviral responses during acute infection. Thus, some HCV infections may
be "silent" because they minimally activate the TLR and/or IFN cellular defense
systems. This would have obvious selective advantage for the virus and could
contribute to the establishment of chronic infection. Alternatively, when a virus
enters cells in a "noisy" fashion, it has a poor chance of establishing chronic infection
because the innate antiviral responses would quickly shut down virus replication.
Finally, stimulation of the IFN system by the HCV core protein may be required
to balance the anti-IFN functions of other HCV proteins such as E2 (Taylor et al.,
1999), NS5A (Gale et al., 1997; Polyak et al., 2001), and NS3 (Foy et al., 2003)
during certain stages of the HCV replication cycle.

EXTRACELLULAR VERSUS INTRACELLULAR EFFECTS OF CORE

HCV core is found within infected cells as well as in patient serum (Kashiwakuma
et al., 1996; Widell et al., 2002). Extracellular core protein likely affects the
modulation of T cell function, TLR signaling and DC function as described above.
Thus, it is important to consider the contribution of extracellular and intracellular
core protein to the biological activity in question. Indeed, CD81 engagement by
the HCV envelope glycoprotein E2 inhibits NK and T cell cytotoxic function
and signal transduction (Crotta et al., 2002; Tseng and Klimpel, 2002; Wack et
al., 2001), and induced pro-inflammatory chemokine expression in hepatocytes
(Balasubramanian et al., 2003). Thus, immune function may be altered as cells
"sample" the microenvironment through HCV-host interactions that are limited to
molecules on the cell surface, such as the HCV core-TLR or core-C1qR interaction.
Moreover, these extracellular HCV-host interactions may also contribute to HCV
pathogenesis.

HCV CORE AND PATHOGENESIS

Recent work has demonstrated that the HCV core protein may also participate in the
pathogenesis of liver disease. Development of fibrosis is characterized histologically
with infiltration of inflammatory lymphocytes, hepatocellular apoptosis, and Kupffer
cell activation. HSC proliferate and undergo and become highly activated, which
involves secretion of large amounts of extracellular matrix proteins (Bataller and
Brenner, 2005). Despite a large body of literature from clinical and animal studies
on fibrosis development, very little is known about how HCV causes fibrosis.

A recent study found that addition of recombinant core protein to activated human
hepatic stellate cells (HSC) stimulated intracellular signaling pathways, while viral
transduction of HCV core into HSCs caused increased cell proliferation (Bataller et
al., 2004). Interestingly, the HSC response appeared to differ between core and other
HCV non-structural proteins. The data suggest that HCV core and non-structural
proteins can modulate the activity of HSC, which may contribute to fibrosis. This
study also reinforces the notion that HCV proteins can have intracellular as well
as extracellular effects on a variety of cells.

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A second important point from the study of Bataller et al., (Bataller and Brenner,
2005), is that HCV proteins including core induce oxidative stress on HSC which is
involved in HSC activation. Indeed, antioxidant therapy reduces the effects of HCV
proteins on HSCs. This finding is in line with the current thinking that oxidative
stress is central to induction of fibrosis in many model systems. HCV core induced
oxidative stress also affects mitochondrial physiology.

HCV CORE AND MITOCHONDRIAL DYSFUNCTION

Expression of HCV core protein in transgenic mice and in cell culture induces
oxidative stress. It has been shown that core protein localizes to mitochondria,
between the mitochondrial outer membrane and ER (Moriya et al., 1998; Moriya
et al., 2001a; Okuda et al., 2002; Schwer et al., 2004; Suzuki et al., 2005), as
described in the targeting section above. Core protein expression and mitochondrial
localization inhibits electron transport at complex I, increases complex I reactive
oxygen species (ROS) production, decreases mitochondrial glutathione, and
increases mitochondrial permeability transition in response to exogenous oxidants
such as alcohol (Korenaga et al., 2005; Okuda et al., 2002; Wen et al., 2004). These
effects are associated with increased hepatocyte apoptosis in the presence of HCV
core protein, ethanol and cytochrome P4502E1. Like the case with HSC, core and
ethanol metabolism effects on apoptosis can be prevented with antioxidants (Otani
et al., 2005).

HEPATITC STEATOSIS AND HEPATOCARCINOGENESIS

Evidence has been accumulating that HCV core protein is directly involved in
pathogenesis (Giannini and Brechot, 2003; McLauchlan, 2000). As shown in Table
1, many cellular proteins, which interact with core protein have been identified.
Several studies have suggested that the core protein plays a crucial role for
hepatocarcinogenesis (Chang et al., 1998; Moriya et al., 1998; Ray et al., 1996).

Recent studies have highlighted steatosis as a basis of HCV-associated HCC (Lerat

et al., 2002; Moriya et al., 1998). Steatosis, which is an accumulation of fat deposits
in hepatocytes, is one of the histological features of chronic hepatitis C (Bach et
al., 1992; Lefkowitch, 2003). In vitro studies have shown that HCV core protein
associates with cellular lipid droplets, via direct interaction with apolipoprotein A2
(Barba et al., 1997; Shi et al., 2002). The mice transgenic for HCV core gene have
been shown to develop steatosis and hepatocellular carcinoma (HCC) (Moriya et al.,
1998; Moriya et al., 1997b). Steatosis in the core-transgenic mice is age-dependent
and characterized by the appearance of micro- and macro-vesicular lipid droplets
(Moriya et al., 1998). Lerat et al. have confirmed that transgenic mice expressing
the whole genome of HCV also develops steatosis and HCC (Lerat et al., 2002).

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Table 1. Cellular proteins that bind to the HCV core protein. The list contains cellular proteins with
various cellular functions that interact with HCV core. The interaction of HCV core with these
cellular proteins may have pathogenic implications. Please refer to the text for details.
Core-Interacting protein Function Reference
Apolipoprotein AII lipid metabolism Sabile et al., 1999; Shi et al., 2002
CAP-Rf RNA helicase You et al., 1999
complement receptor gC1qR T-cell response Kittlesen et al., 2000
cyclin-dependent kinase 7 cell cycle Ohkawa et al., 2004
DEAD box protein RNA helicase Mamiya and Worman, 1999
DEAD box protein 3 RNA helicase Owsianka and Patel, 1999
heterogeneous nuclear
ribonucleoprotein K transcriptional control Hsieh et al., 1998
JAK1/2 signal transduction Hosui et al., 2003
lymphotoxin-β receptor cytotoxicity Chen et al., 1997
p53 transcriptional control Otsuka et al., 2000
p73 transcriptional control Alisi et al., 2003
proteasome activator PA28γ protein stability Moriishi et al., 2003
retinoid X receptor α transcriptional control Tsutsumi et al., 2002
Smad3 transcriptional control Cheng et al., 2004
Sp110b transcriptional control Watashi et al., 2003
STAT3 cell transformation Yoshida et al., 2002
TAFII28 transcriptional control Otsuka et al., 2000
Tumor necrosis factor receptor 1 apoptosis Zhu et al., 2001
14-3-3 protein signal transduction Aoki et al., 2000

Although the molecular mechanisms of steatosis caused by the core protein is still
unclear, the core protein may alter lipid metabolism by interacting with cellular
proteins involved in lipid accumulation and storage in hepatocytes (Barba et
al., 1997; Sabile et al., 1999; Shi et al., 2002). The concentration of carbon 18
monosaturated fatty acids were increased in the livers of the core-transgenic mice
and chronic hepatitis C patients, suggesting that HCV core affects a specific pathway
in lipid metabolism (Moriya et al., 2001b). Nonetheless, transgenic mouse lines
established by other groups did not show either steatosis nor HCC (Kawamura et
al., 1997; Pasquinelli et al., 1997). These discrepancies suggest that not only the
viral proteins but also other factors are involved in hepatocarcinogenesis. These
discrepancies may be due to differences in genetic backgrounds of the mice and
expression levels of the viral proteins.

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ACKNOWLEDGEMENTS
SJP is partially supported by NIH grants AA13301 and DK62187, and the University
of Washington Royalty Research Fund. JRL received support from Puget Sound
Partners, and KCK received support from NIH training grant T32 CA09229.
IS and TM are supported in part by grants from the Program for Promotion of
Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief,
RandD Promotion and Product Review of Japan (ID:01-3) and the Second Term
Comprehensive 10-year Strategy for Cancer Control of the Ministry of Health,
Labor, and Welfare of Japan. IS and TM also thank their colleagues, T.Tsutsumi,
K.Ishii, H.Aizaki, K. Murakami, R.Suzuki, T, Suzuki, (National Institute of
Infectious Diseases), Y.Shintani, H.Fujie, K. Moriya, K.Koike, (Tokyo University)
and K.Moriishi, Y. Matsuura (Osaka University) for contribution.

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Chapter 4

HCV Glycoproteins: Assembly of a

Functional E1-E2 Heterodimer
Muriel Lavie, Anne Goffard and Jean Dubuisson

ABSTRACT
The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein
by signal peptidase cleavages. These glycoproteins are type I transmembrane
proteins with a highly glycosylated N-terminal ectodomain and a C-terminal
hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate
as a noncovalent heterodimer. The transmembrane domains of HCV envelope
glycoproteins play a major role in E1-E2 heterodimer assembly and subcellular
localization. The envelope glycoprotein complex E1-E2 has been proposed to
be essential for HCV entry. However, for a long time, HCV entry studies have
been limited by the lack of a robust cell culture system for HCV replication and
viral particle production. Recently, a model mimicking the entry process of HCV
lifecycle has been developed by pseudotyping retroviral particles with native HCV
envelope glycoproteins, allowing the characterization of functional E1-E2 envelope
glycoproteins. Here, we review our understanding to date on the assembly of the
functional HCV glycoprotein heterodimer.

INTRODUCTION
As obligate intracellular parasites, all viruses have evolved ways of entering
target cells to initiate replication and infection. The first step in virus entry is the
recognition of host cells through cell surface receptor(s). This initial engagement
can mediate attachment as well as act as a primer for subsequent conformational
alteration, leading to virus entry into host cell. In many cases, interaction with a
receptor is important for defining the tropism of a virus for a particular organism,
tissue or cell type. Enveloped viruses possess a lipid bilayer that surrounds their
nucleocapsid. The glycoproteins present in their envelope are involved in the
receptor-binding step. After attachment, the entry of these viruses into cells requires
the fusion of the viral and a cellular membrane by a process that is also driven by the
viral envelope glycoproteins. To fulfill these functions, viral envelope glycoproteins
have to adopt dramatically different conformations during the virus lifecycle. In
addition, these conformational changes have to occur at a precise time of the virus
lifecycle, and thus, have to be tightly modulated.

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HCV encodes two envelope glycoproteins, named E1 and E2. For a long time,
the lack of a cell culture system supporting efficient HCV replication and particle
assembly has hampered the characterization of the envelope proteins present on
the virion. Cell culture transient expression systems have allowed investigators
to characterize the first steps in the biogenesis of HCV envelope glycoproteins
(reviewed in: Op De Beeck et al., 2001). In addition, surrogate models have also
been developed to study the entry steps of HCV lifecycle (reviewed in: Op De
Beeck and Dubuisson, 2003). However, it is only recently that a model mimicking
the entry process of HCV lifecycle has been developed. This has been achieved
by pseudotyping retroviral particles with native HCV envelope glycoproteins
(Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). This new tool
allows, for the first time, the characterization of the assembly of functional HCV
envelope glycoproteins.

BIOGENESIS OF HCV ENVELOPE GLYCOPROTEINS

CLEAVAGE OF HCV GLYCOPROTEINS FROM THE VIRAL POLYPROTEIN

As for the other members of the Flaviviridae family, the genome of HCV encodes
a single polyprotein. This ~3010 amino acid polyprotein is processed by cellular
(signal peptidase and signal peptide peptidase) and viral proteases (NS2-3 and
NS3-4A) to generate at least 10 polypeptides (reviewed in: Penin et al., 2004). The
nonstructural proteins are released from the polyprotein after cleavage by HCV
proteases NS2-3 and NS3-4A, whereas the structural proteins are released by host
endoplasmic reticulum (ER) signal peptidase(s) (Fig. 1)(reviewed in Reed and Rice,
2000). Further processing mediated by a signal peptide peptidase also occurs at the
C-terminus of the capsid protein (McLauchlan et al., 2002). Most cleavages in the
polyprotein precursor proceed to completion during or immediately after translation
(Grakoui et al., 1993; Dubuisson et al., 1994; Lin et al., 1994; Mizushima et al.,
1994; Dubuisson and Rice, 1996). Partial cleavages occur at the E2/p7 and p7/NS2
sites, leading to the production of an uncleaved E2p7NS2 molecule. While most of
NS2 is progressively cleaved from the E2p7NS2 precursor, the cleavage between
E2 and p7 does not change over time, at least for most HCV strains analyzed
(Dubuisson, 2000). Thus, this results in cleavage products consisting of E2, E2p7,
p7, and NS2.

The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage

sites can efficiently function as signal peptides. Indeed, when fused to a reporter
protein, the signal peptides of p7 and NS2 are efficiently cleaved (Carrère-Kremer
et al., 2004). These data indicate that inefficiency of cleavage at E2/p7 and p7/NS2
sites is not due to the presence of suboptimal signal peptides. The p7 polypeptide is
a polytopic membrane protein containing two transmembrane domains with both
its N- and C-termini oriented toward the ER lumen (Fig. 1)(Carrère-Kremer et al.,

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Fig. 1. Processing of the N-terminal one-third of HCV polyprotein. The arrows show host signal
peptidase cleavages. Partial cleavages at E2/p7 and p7/NS2 sites are indicated by dotted arrows.
Cleavage by the host cell signal peptide peptidase (SPP) is indicated by scissors. The signal peptide
and signals of reinitiation of translocation are shown as a black cylinder and light grey cylinders,
respectively. The transmembrane domains of HCV envelope glycoproteins are represented in their
pre-cleavage topology. Post-cleavage reorientation of the glycoprotein signals of reinitiation of
translocation is indicated by curved arrows.

2002). Interestingly, the presence of the first transmembrane domain of p7 reduces

the efficiency of p7/NS2 cleavage (Carrère-Kremer et al., 2004). Sequence analyses
and mutagenesis studies have also identified structural determinants responsible for
the partial cleavage at both E2/p7 and p7/NS2 sites (Carrère-Kremer et al., 2004).
In addition, the short distance between the cleavage site of E2/p7 or p7/NS2 and
the predicted transmembrane α-helix located downstream of the cleavage sites
might impose additional structural constraints to these cleavage sites (Fig. 1). Such
constraints in the processing of a polyprotein precursor are likely essential for HCV
to post-translationally regulate the kinetics and/or the level of expression of p7 as
well as NS2 and E2 mature proteins.

The processing at the E2p7 site has been further explored. It has been reported to
be more efficient in genotype 1b (strain BK) than in the genotype 1a (strain H77c)
(Dubuisson et al., 1994; Lin et al., 1994). A sequence comparison of p7 signal
peptides of these two viral strains has identified a difference of 3 amino acids
and mutational analysis has shown that the V720L change in the H77c sequence
substantially increases the efficiency of processing at the E2/p7 site (Isherwood and
Patel, 2005). Although, when expressed alone, p7 protein has been shown to adopt
a double membrane spanning topology with both extremities orientated luminally in
the ER (Carrère-Kremer et al., 2002), the C-terminal part of E2p7 proteins has been
found to be located in the cytosol (Isherwood and Patel, 2005). These data suggest

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that p7 can potentially adopt a dual transmembrane topology. It remains, however,

to be shown whether an E2p7 with a cytosolic orientation of the C-terminus of p7
exists when this protein is expressed in the context of the polyprotein.

Since p7 and NS2 are not essential for HCV genomic replication (Lohmann et
al., 1999; Blight et al., 2000), they will likely play their role in virion assembly, a
process that is supposed to be tightly regulated. It has recently been shown that p7
reconstituted into artificial lipid membranes homo-oligomerizes and behaves as an
ion channel protein (Griffin et al., 2003; Pavlovic et al., 2003; Premkumar et al.,
2004). It is likely that, when bound to E2, p7 cannot oligomerize and function as
an ion channel, and the existence of E2p7 would therefore reduce the amount of
functional p7 molecules available. Production of precursors like E2p7NS2 and E2p7
might be a means to maintain p7 inactive during the phase of the accumulation of
E2 molecules required for HCV envelope formation. Alternatively, such precursors
might also control the temporal release of E2 and NS2.

GLYCOSYLATION OF HCV ENVELOPE GLYCOPROTEINS

N-linked glycosylation is one of the most common types of protein modification,
and it occurs by the transfer of an oligosaccharide from a lipid intermediate to an
Asn residue in the consensus sequence Asn-X-Thr/Ser of a nascent protein, where X
is any amino acid except Pro (Kornfeld and Kornfeld, 1985; Gavel and von Heijne,
1990). The addition of this glycan is catalyzed by the oligosaccharyltransferase,
which is closely associated with the translocon through which the nascent peptidic
chains emerge in the ER lumen (Silberstein and Gilmore, 1996). However, not
every tripeptide sequence in a protein sequence is used for carbohydrate addition
(Gavel and von Heijne, 1990). In the early secretory pathway, the glycans play a
role in protein folding, quality control and certain sorting events. Viral envelope
proteins usually contain N-linked glycans that can play a major role in their folding,
in their entry functions or in modulating the immune response (Hebert et al., 1997;
Ohuchi et al., 1997a; Ohuchi et al., 1997b; van Kooyk and Geijtenbeek, 2003; von
Messling and Cattaneo, 2003; Wei et al., 2003).

The ectodomains of HCV envelope glycoproteins E1 and E2 are highly modified

by N-linked glycans. E1 and E2 possess up to 6 and 11 potential glycosylation
sites, respectively (Fig. 2). Sequence analyses of E1 indicate that 5 potential N-
glycosylation sites are strongly conserved among HCV genotypes (Goffard and
Dubuisson, 2003; Zhang et al., 2004b). However, in one case the presence of a
proline residue immediately downstream the glycosylation site is unfavorable
for glycosylation, and it has been confirmed experimentally that this site is not
glycosylated (Meunier et al., 1999). Interestingly, the glycosylation site of E1 at
position 250 is poorly conserved; this site is indeed only observed in genotypes
1b and 6 (Fig. 2)(Goffard and Dubuisson, 2003). Most E2 glycosylation sites are

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Fig. 2. Schematic representation of E1 and E2 features. Positions of N-linked glycans are indicated as
an N followed by a number related to the relative position of the potential glycosylation site in each
glycoprotein. The numbers correspond to the positions in the polyprotein of reference strain H (acc.
Number AF009606). Glycans involved in HCVpp entry are indicated with a black square (Goffard
et al., 2005). Glycosylation sites for which the mutation alters E1E2 folding are indicated with a grey
circle (Goffard et al., 2005). The hypervariable region 1 (HVR1) of E2 is shown as a grey box. The
black boxes correspond to E2 epitopes recognized by neutralizing antibodies (Hsu et al., 2003). The
sequences of the transmembrane domains of HCV envelope glycoproteins are indicated above their
corresponding region in E1 and E2. The two hydrophobic segments in these regions are underlined.
The charged residues present between the two hydrophobic stretches are in white lettering. Arrows
indicate the positions of inserted alanine residues that disrupt HCV E1E2 heterodimerization (Op
De Beeck et al., 2000).

also well conserved. Indeed, global sequence analyses of potential glycosylation

sites in E2 indicate that nine of the eleven sites are strongly conserved (Goffard
and Dubuisson, 2003; Zhang et al., 2004b). The two remaining sites, N5 and N7,
show conservation levels of 75% and 89%, respectively (Goffard and Dubuisson,
2003).

Mutants of E1 and E2 have been produced to characterize the glycosylation of these

proteins (Meunier et al., 1999; Nakano et al., 1999; Slater-Handshy et al., 2004;
Goffard et al., 2005). In the context of the H strain, the 4 potential glycosylation
sites of E1 were shown to be occupied by glycans (Meunier et al., 1999; Goffard
et al., 2005). In the case of E2, a first study has shown that mutation of some
glycosylation sites in the context of a truncated form of E2 alters the recognition
by sera from HCV patients (Nakano et al., 1999); however, these mutants were not
characterized in terms of glycosylation and no clear conclusion can be drawn from
this study. More recently, glycosylation mutants have been produced in the context
of a truncated form of E2 ending at position 660 (Slater-Handshy et al., 2004). The
E2 sequence of HCV isolate used in this study contains 10 instead of 11 potential

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glycosylation sites, the site N5 at position 476 being missing (Fig. 2). Interestingly,
the last two glycosylation sites, N10 and N11, were not occupied in E2660 (Slater-
Handshy et al., 2004). However, at least one of these sites was occupied in the
context of full-length E2. A more recent mutagenesis study, in the context of an
E2 glycoprotein containing 11 potential glycosylation sites, has shown that all the
sites are occupied by glycans (Goffard et al., 2005). In this case, E2 was expressed
as a polyprotein containing full-length E1 and E2. Altogether, these data indicate
that full-length and truncated forms of E2 can have different properties.

The addition of the glycan precursor is catalyzed by the oligosaccharyltransferase,

and this enzyme is thought to have access only to nascent chains as they emerge from
the ribosome at the luminal face of the rough ER (Silberstein and Gilmore, 1996).
The glycosylation process of HCV envelope glycoprotein E1 has been analyzed in
the context of a Man-P-Dol-deficient cell line (B3F7) and it has been shown to occur
post-translationally (Duvet et al., 2002), indicating that the oligosaccharyltransferase
has also access to the E1 glycoprotein for more than an hour after its translation. A
characterization of HCV glycoprotein E1 has also shown that, in the absence of E2,
different glycoforms of E1 are produced and the glycosylation of E1 is improved
by co-expression of E2 in cis (Dubuisson et al., 2000).

FOLDING OF HCV ENVELOPE GLYCOPROTEINS

HCV envelope glycoproteins have been shown to assemble as a noncovalent
E1E2 heterodimer (Deleersnyder et al., 1997). However, at least in heterologous
expression systems, HCV envelope glycoproteins have a tendency to also form
misfolded aggregates stabilized by disulfide bonds (reviewed in (Dubuisson,
2000)). Analyses of HCV envelope glycoproteins with conformation-sensitive
antibodies are therefore necessary to discriminate noncovalent heterodimers
from misfolded complexes (Deleersnyder et al., 1997; Cocquerel et al., 2003b).
Alternatively, such discrimination can also be made by analyzing disulfide-bond
formation by migrating HCV envelope glycoproteins on SDS-PAGE under non-
reducing conditions (Dubuisson and Rice, 1996; Brazzoli et al., 2005). Analyses
of the formation of conformation-dependent epitopes and disulfide-bond formation
indicate that folding of HCV envelope glycoproteins is a slow process (Deleersnyder
et al., 1997; Dubuisson and Rice, 1996; Duvet et al., 1998; Brazzoli et al., 2005).
Interestingly, the folding of E1 has been shown to be dependent on the co-expression
of E2 (Michalak et al., 1997; Patel et al., 2001). In addition, it has also been shown
that the folding of E2 is also dependent on the co-expression of E1 (Cocquerel et
al., 2003a; Brazzoli et al., 2005). Altogether, these observations indicate that HCV
envelope glycoproteins cooperate for the formation of a functional complex. These
observations also indicate that, although some degree of folding can be observed
in E2 expressed alone (Michalak et al., 1997; Cocquerel et al., 2003a), both
glycoproteins need to be co-expressed to analyze their functional properties.

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During their folding, HCV envelope glycoproteins have been shown to interact
with calnexin (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al.,
2001; Brazzoli et al., 2005), a lectin-like ER chaperone, which shows an affinity for
monoglucosylated N-linked oligosaccharides (Trombetta and Helenius, 1998). Both
E1 and E2 have been found to associate rapidly with calnexin and dissociate slowly,
suggesting a role of this chaperone in the folding of HCV envelope glycoproteins
(Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001). However,
more recent data suggest that only E1 interacts with calnexin (Brazzoli et al., 2005).
Differences in the cell lines used and/or in the levels of expression of the envelope
glycoproteins might potentially explain these discrepancies. Further experiments
in cell cultures infected with native HCV particles will be needed to confirm the
involvement of calnexin in the folding E2.

The presence of glycans on HCV envelope glycoproteins can potentially affect

their folding either directly or through interaction with calnexin. Site-directed
mutagenesis studies have indeed shown that the absence of some glycans in E1 (N1
and N4) and E2 (N8 and N10) leads to misfolding of HCV envelope glycoproteins
(Fig. 2)(Meunier et al., 1999; Goffard et al., 2005). This alteration in folding was
not due to the lack of interaction of HCV envelope glycoproteins with calnexin,
suggesting that the mutations would rather have a direct effect on protein folding.
The presence of a large polar saccharide is indeed known to affect the folding at least
locally by orienting polypeptide segments toward the surface of protein domains
(Imperiali and O'Connor, 1999; Wormald and Dwek, 1999).

INVOLVEMENT OF THE TRANSMEMBRANE DOMAINS IN THE BIOGENESIS

OF E1E2 HETERODIMER

MEMBRANE ANCHOR AND SIGNAL SEQUENCE

Due to their resistance to alkaline or salt extraction, HCV envelope glycoproteins
have been confirmed to be membrane associated proteins (Ralston et al., 1993;
Cocquerel et al., 2001). In addition, deletion of the C-terminal hydrophobic regions
of these proteins leads to their secretion, indicating that these regions are involved in
membrane anchoring (Michalak et al., 1997). Sequence analysis of a large number
of HCV isolates has shown that the C-termini of E1 and E2 contain hydrophobic
sequences that are less than 30 amino acid residues long (Fig. 2)(Cocquerel et al.,
2000). As in other viruses of the Flaviviridae family, these regions are composed
of two stretches of hydrophobic residues separated by a short segment containing
at least one fully conserved positively charged residue (Cocquerel et al., 2000).
Interestingly, when fused to a reporter protein the second hydrophobic stretch
functions as a signal sequence (Cocquerel et al., 2002), which is in agreement with
the observation that HCV envelope glycoproteins are released from the polyprotein
precursor after cleavage by host signal peptidase(s) (Dubuisson et al., 2002). It is
worth noting that in the context of HCV polyprotein, only the sequence located at

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the C-terminus of the immature form of the capsid protein is a true signal peptide
that will interact with the signal recognition particle (Santolini et al., 1994). The
sequence present at the C-terminus of E1 and E2 do not interact with the signal
recognition particle, and they should be called signals of reinitiation of translocation
(Fig. 1). Deletion of these signals leads to the secretion of E1 and E2, indicating that
these signals are involved in their membrane anchoring (Cocquerel et al., 2000).

ER RETENTION FUNCTION
HCV envelope glycoproteins are retained in the ER (Dubuisson et al., 1994;
Deleersnyder et al., 1997; Duvet et al., 1998), and ER retention signals are present
in the transmembrane domains of E1 and E2 (Cocquerel et al., 1998; Cocquerel
et al., 1999). In addition, the charged residues of the transmembrane domains
of E1 (Lys) and E2 (Asp and Arg) play a key role in the ER retention of these
glycoproteins (Cocquerel et al., 2000). It has been proposed that an additional
ER retention signal might also be present in the ectodomain of E1 (Mottola et al.,
2000). Interestingly, in some conditions of overexpression a small fraction of HCV
envelope glycoproteins has been shown to accumulate at the plasma membrane
(Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et
al., 2004). Cell surface expression of E1 and E2 is likely due to the accumulation
of small amounts of glycoproteins escaping the ER-retention machinery, due to
saturation of this mechanism.

ROLE IN HETERODIMERIZATION
In addition to their anchoring, signal sequence and ER retention functions, the
transmembrane domains of HCV envelope glycoproteins have also been shown
to play a major role in the assembly of E1E2 heterodimer. Indeed, deletion of the
transmembrane domain of E2 or its replacement by the anchor signal of another
protein abolishes the formation of E1E2 heterodimer (Selby et al., 1994; Michalak
et al., 1997; Cocquerel et al., 1998; Patel et al., 2001). Other studies by site-directed
mutagenesis or alanine scanning insertion mutagenesis (Cocquerel et al., 2000;
Op De Beeck et al., 2000) have confirmed that the transmembrane domains of E1
and E2 play a direct role in E1E2 assembly. In addition, alanine scanning insertion
mutagenesis allowed to identify two distinct segments in the transmembrane domain
of E1 and one in the transmembrane domain of E2 that were specifically involved
in E1E2 assembly (Fig. 2). Interestingly, at least one region located outside of the
transmembrane domains has also been shown to be involved in heterodimerization
(Drummer and Poumbourios, 2004).

TOPOLOGICAL CHANGE IN THE TRANSMEMBRANE DOMAIN OF HCV GLYCOPROTEINS

The topology of the transmembrane domain of HCV envelope glycoproteins has
given rise to some controversy. Indeed the presence of a first hydrophobic stretch
and a signal sequence function separated by charged residues in the transmembrane
domains of E1 and E2 has suggested that they might be composed of two membrane

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spanning segments with the charged residues facing the cytosol (Charloteaux et
al., 2002). This type of organization has been observed in the C-terminal region
of the envelope glycoprotein E2 of the alphaviruses as well as for the envelope
proteins of the flaviviruses (Strauss and Strauss, 1994; Op De Beeck et al., 2003;
Zhang et al., 2003). However sequence analysis and data of alanine scanning
insertion mutagenesis were in favor of a single spanning topology of E1 and E2
transmembrane domain (Cocquerel et al., 2000; Op De Beeck et al., 2000). A study
of the topology of the transmembrane domains of HCV envelope proteins has been
performed by determining the accessibility of their N- and C-termini in selectively
permeabilized cells (Cocquerel et al., 2002). This work has shown that before signal
sequence cleavage at their C-terminus, the transmembrane domains form a hairpin
structure (Fig. 1). However, after cleavage between E1 and E2 or between E2 and
p7, the second C-terminal hydrophobic stretch is reoriented towards the cytosol,
leading to the formation of a single membrane-spanning domain. Here again, the
charged residues located in the middle of the transmembrane domains were shown
to play a crucial role in their structural dynamics (Cocquerel et al., 2002).

ROLE OF HCV ENVELOPE GLYCOPROTEINS IN VIRUS ENTRY

For most viruses, entry into the cytosol is a multistep process, during which the
host cell assists the incoming virus. Viruses first attach themselves to components
of the plasma membrane, which they use as non-specific attachment factors or as
specific cell surface receptors. Viral attachment is mediated by the binding of a
protein present at the surface of the virion to a molecule on the cell surface acting
as a virus receptor. The envelope glycoprotein complex E1E2 is the viral component
thought to be present at the surface of HCV particles and it is therefore the obvious
candidate ligand for cellular receptors. Receptor binding can activate cellular
endocytic pathways through which viruses are internalized in endosomes. When
they reach the appropriate intracellular location, viruses are activated for penetration
by cellular signals and make their way through the membrane of the endosome, or
through the plasma membrane for those that do not enter by endocytosis. Enveloped
viruses fuse their lipid envelope with the plasma membrane or the membrane of an
endosome, resulting in the release of the nucleocapsid into the cytosol.

MODELS TO STUDY HCV ENTRY

In the absence of a robust cell culture system to amplify HCV, several models have
been developed to study HCV entry. In a first approach, a soluble form of HCV
glycoprotein E2 has been used to identify cell surface proteins potentially involved
in HCV entry (Rosa et al., 1996; Pileri et al., 1998). Although this approach is
potentially interesting in protein-protein interactions studies, it cannot be used
to study the entire entry process. In addition, as discussed above, due to their
cooperative role in folding, both glycoproteins need to be co-expressed to analyze
their functional properties. To study the role of E1E2 envelope glycoproteins in HCV

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entry, several surrogate models of HCV particles have therefore been developed.
As a first approach, virus-like particles have been produced in insect cells infected
by a recombinant baculovirus containing the cDNA of HCV structural proteins
(Baumert et al., 1998). However these particles are not infectious and they are
retained in an intracellular compartment. It is therefore difficult to evaluate how
close these virus-like particles are to native virion. In addition, due to the absence
of infectivity, these particles cannot be used to study the fusion process. Another
approach to study HCV entry has been to produce virosomes by incorporating E1E2
heterodimers into liposomes (Lambot et al., 2002). These virosomes can be used
to study the interactions between E1E2 heterodimers and cell surface receptors.
However, it has not been shown whether the envelope glycoproteins incorporated
into these liposomes can induce fusion.

Other models have been based on pseudotyping of viral vectors. The first model that
has been developed was based on vesicular stomatitis virus (VSV) pseudotyped with
modified E1 and/or E2 glycoproteins (Lagging et al., 1998; Matsuura et al., 2001).
In these particles, the transmembrane domains of HCV envelope glycoproteins
have been replaced by the transmembrane domain and cytoplasmic tail of the VSV
envelope glycoprotein G. This allows the export of HCV envelope glycoproteins
to the cell surface (Takikawa et al., 2000). However, some doubts have been raised
on the infectivity of such VSV pseudotyped particles (Buonocore et al., 2002). In
addition, replacement of HCV envelope glycoproteins has been shown to alter their
entry function (Hsu et al., 2003).

More recently, retroviruses have also been used to produce pseudotyped particles
containing HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al.,
2003; Hsu et al., 2003). Murine leukemia virus (MLV) or human immunodeficiency
virus (HIV) vectors were used. Retroviruses are indeed well known to be able to
incorporate in their envelope a variety of cellular and viral glycoproteins (Ott,
1997; Sandrin et al., 2002). In addition, they can easily package and integrate
genetic markers into host cell DNA (Negre et al., 2002). All these properties were
exploited to produce viral pseudoparticles expressing E1E2 at their surface and
packaging a reporter gene that allows to monitor viral infection of the target cell.
HCV pseudoparticles (HCVpp) are produced by transfecting 293T cells with three
expression vectors encoding the E1E2 polyprotein, the retroviral core proteins and
a packaging-competent retrovirus-derived genome containing a marker gene (Fig.
3). Because MLV and HIV are supposed to assemble at the plasma membrane and
HCV glycoproteins are retained in the ER, a first approach has been to modify the
transmembrane domains of E1 and E2 to re-address them at the plasma membrane
(Hsu et al., 2003; Pohlmann et al., 2003). However pseudoparticles bearing
such modified HCV envelope glycoproteins were not infectious. Surprisingly,
in the absence of any modification of HCV envelope glycoproteins, infectious

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Fig. 3. Production of HCV pseudoparticules (HCVpp). For the production of HCVpp, human embryo
kidney cells 293T are transfected with three expression vectors. The first vector encodes retroviral
Gag and Pol proteins. Gag proteins are responsible for particle budding at the plasma membrane and
RNA encapsidation via recognition of the specific retroviral encapsidation sequence (ψ). The second
vector harbors a ψ sequence for encapsidation and encodes a reporter protein (Luciferase). This vector
also contains retroviral sequences that are necessary for the reverse transcription of genomic RNA
into proviral DNA and for integration of the proviral DNA in the host genomic DNA by the retroviral
protein Pol encoded by the first vector. The third vector encodes HCV envelope glycoproteins, which
are responsible for the cell tropism and fusion of HCVpp with the target cell membrane. HCVpp
contain Gag, Pol, E1 and E2 proteins as well as the RNA encoding the luciferase protein. Infectivity
of HCVpp is evaluated by measuring the amount of luciferase expressed in target cells.

pseudoparticles were produced (Bartosch et al., 2003b; Drummer et al., 2003;

Hsu et al., 2003). Interestingly, due to saturation of the ER retention machinery,
the cells used to produce HCVpp were shown to express a small fraction of HCV

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envelope glycoproteins at the plasma membrane (Bartosch et al., 2003b; Drummer

et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). This accumulation at the
plasma membrane might therefore be sufficient to incorporate native HCV envelope
glycoproteins into retroviral pseudotyped particles.

The data that have been accumulated on these pseudoparticles strongly suggest that
they mimic the early steps of HCV infection. Indeed, they exhibit a preferential
tropism for hepatic cells and they are specifically neutralized by anti-E2 monoclonal
antibodies as well as sera from HCV-infected patients (Bartosch et al., 2003b;
Hsu et al., 2003; Op De Beeck et al., 2004). These HCVpp therefore represent the
best tool available to study functional HCV envelope glycoproteins. An analysis
of the glycoproteins associated with HCVpp has shown the heterogeneous nature
of E1 and E2 incorporated into HCVpp (Flint et al., 2004). This highlights the
difficulty in identifying forms of the HCV glycoproteins that initiate infection.
However, characterization of HCVpp envelope glycoproteins with conformation-
sensitive neutralizing monoclonal antibodies has shown that the functional unit
is a noncovalent E1E2 heterodimer (Op De Beeck et al., 2004). In addition,
coexpression of both envelope glycoproteins has been shown to be necessary to
produce infectious pseudoparticles (Bartosch et al., 2003b), confirming that only
the E1E2 heterodimer is functional.

HCV RECEPTORS
As a first approach to identify potential HCV receptor(s), a soluble form of HCV
glycoprotein E2 has been used. This allowed to identify the CD81 tetraspanin (Levy
and Shoham, 2005) as a putative receptor for HCV (Pileri et al., 1998). A very similar
approach identified the scavenger receptor class B type I (SR-BI) (Scarselli et al.,
2002), a high-density lipoprotein (HDL)-binding molecule (Connelly and Williams,
2004), and the mannose binding lectins DC-SIGN and L-SIGN (van Kooyk and
Geijtenbeek, 2003) as additional candidate receptors for HCV (Gardner et al., 2003;
Lozach et al., 2003; Pohlmann et al., 2003; Ludwig et al., 2004). Heparan sulfate
has also been shown to interact with HCV glycoprotein E2, suggesting that this
type of molecule can play a role in HCV entry (Barth et al., 2003). An approach
using virus-like particles produced in insect cells has led to the identification of
the asialoglycoprotein receptor as another candidate receptor for HCV (Saunier
et al., 2003). Finally, because of the physical association of HCV with low- or
very-low-density lipoproteins (LDL or VLDL) in serum, the LDL receptor has
also been proposed as another candidate receptor for HCV. (Agnello et al., 1999;
Monazahian et al., 1999).

A number of cell-surface molecules bind viral envelope glycoproteins without

mediating entry, and validation of a viral receptor or co-receptor requires proof
that the putative receptor is necessary for infection. This is not easy for HCV due

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to the absence of a robust cell culture system to amplify this virus. The recent
development of HCVpp has allowed to further investigate the role of candidate
receptors in virus entry. Among all the candidate receptors, only CD81 and SR-BI
have been shown to play a direct role in HCVpp entry. Indeed, antibodies directed
against CD81 or SR-BI as well as siRNA targeting these receptors reduce HCVpp
infectivity (Bartosch et al., 2003b; Bartosch et al., 2003c; Hsu et al., 2003; Cormier
et al., 2004b; Zhang et al., 2004a; Lavillette et al., 2005b). A soluble domain of
CD81 is also able to compete with HCVpp infectivity (Bartosch et al., 2003b; Hsu
et al., 2003). In addition, HDL, the natural ligands of SR-BI, are able to markedly
enhance HCVpp entry (Meunier et al., 2005; Voisset et al., 2005). This HDL-
mediated enhancement of HCVpp entry involves a complex interplay between
SR-BI, HDL and HCV envelope glycoproteins (Voisset et al., 2005). Interestingly,
the involvement of CD81 and SR-BI in HCVpp entry seems to be conserved among
all the HCV genotypes (McKeating et al., 2004; Lavillette et al., 2005b).

Interactions between viral envelope glycoproteins and potential receptors can have
other consequences than virus entry. It has been shown that intracellular interaction
between HCV envelope glycoproteins and CD81 can lead to secretion of exosomes
containing E1 and E2 glycoproteins (Masciopinto et al., 2004). Interestingly, a
soluble form of E2 is also able to bind CD81 at the surface of natural killer cells,
and this interaction inhibits cytotoxicity and cytokine production by these cells
(Crotta et al., 2002; Tseng and Klimpel, 2002). Binding of a soluble form of E2
can also provide a co-stimulatory signal for T cells (Wack et al., 2001; Soldaini et
al., 2003;) and up-regulate matrix metalloproteinase-2 in human hepatic stellate
cells (Mazzocca et al., 2005). It remains however to be determined whether HCV
glycoprotein expressed in the context of native particles will have the same effects
on cell functions.

HCVpp have also been used to investigate the role of other candidate receptors in
HCV entry. HCVpp as well as native HCV particles have been shown to bind to
cells expressing L-SIGN and DC-SIGN (Gardner et al., 2003; Pohlmann et al., 2003;
Lozach et al., 2004). Although these molecules are not expressed on hepatocytes,
HCV interactions with L-SIGN and DC-SIGN may contribute to establishment or
persistence of infection both by the capture and delivery of virus to the liver and
by modulating dendritic cell functions as recently suggested (Cormier et al., 2004a;
Lozach et al., 2004). Finally, there is no clear evidence that the LDL receptor is a
major receptor for HCVpp (Bartosch et al., 2003b).

Interestingly, all the cells permissive to HCVpp co-express CD81 and SR-BI and
are of liver origin (Bartosch et al., 2003c; Hsu et al., 2003; Zhang et al., 2004a).
However, there are some other cell lines coexpressing CD81 and SR-BI that are
non-permissive to infection and which are of non-hepatic origin. These results

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suggest that additional molecule(s), expressed in hepatic cells only, are necessary
for HCV entry. Further investigations with HCVpp should allow to identify such
molecule(s).

FUNCTIONAL REGIONS OF HCV ENVELOPE GLYCOPROTEINS

HCVpp have been used to investigate the functional role of some regions of HCV
envelope glycoproteins in virus entry. Mutagenesis studies of the transmembrane
domains of HCV envelope glycoproteins have shown that some mutations
can affect the entry function of HCVpp without alteration in the biogenesis of
E1E2 heterodimer and their incorporation into HCVpp (Ciczora Y, Callens N,
Montpellier C, Bartosch B, Cosset FL, Op De Beeck A, Dubuisson J, unpublished
data). This suggests that in addition to their role in E1E2 heterodimerization, the
transmembrane domains of HCV glycoproteins might play a role in coordinating
protein reorganization for the fusion process to occur.

Studies of E2-CD81 interactions and identification of epitopes recognized by

antibodies that inhibit these interactions suggest that the CD81-binding region
consists of discrete segments of E2 that are rearranged within the same domain
during E2 folding (Flint et al., 1999a; Forns et al., 2000a; Yagnik et al., 2000;
Owsianka et al., 2001; Clayton et al., 2002; Hsu et al., 2003). Besides this putative
binding region, the hypervariable region 1 (HVR1)(Weiner et al., 1991), a 27-amino
acid long segment found at the N-terminus of E2 (Fig. 2), has also been suggested
to play a role in cell attachment (Penin et al., 2001; Scarselli et al., 2002). This
region evolves rapidly in infected individuals, suggesting that it is under strong
immune pressure (reviewed in Mondelli et al., 2003). Although an HCV clone
lacking HVR1 was shown to be infectious in chimpanzee, this mutant virus was
attenuated, suggesting that HVR1 plays a facilitating role in HCV infectivity (Forns
et al., 2000b). In addition, deletion of HVR1 reduces HCVpp infectivity (Bartosch
et al., 2003c) and abolishes HDL-mediated enhancement of HCVpp infectivity
(Voisset et al., 2005). Despite strong amino acid sequence variability related to
strong pressure towards change, the chemicophysical properties and conformation
of HVR1 are highly conserved, and HVR1 is a globally basic stretch, with basic
residues located at specific sequence positions. Functional studies of HCVpp
containing mutations in HVR1 indicate that infectivity increases with the number
of basic residues in HVR1 (Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset
FL, Pawlotsky JM, Penin F, Dubuisson J, unpublished data). In addition, a shift in
position of some charged residues modulates infectivity. These data suggest that
HVR1 is a region involved in interaction with a host molecule involved in HCV
entry. However, it remains to be determined whether SR-BI or another putative
receptor is involved in this interaction.

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HCV envelope glycoproteins are highly glycosylated and some maturation of

these glycans has been observed on HCV envelope glycoproteins associated with
HCVpp (Flint et al., 2004; Lozach et al., 2004; Op De Beeck et al., 2004). Mutation
of some glycosylation sites in HCV envelope glycoproteins can reduce or abolish
HCVpp infectivity without apparently affecting folding and incorporation of the
glycoproteins into the particles (Goffard et al., 2005). N-linked glycans at position
N2 and N4 of E2 have indeed been shown to be essential for the entry functions of
HCV envelope glycoproteins (Fig. 2). In addition, some other glycans (N2 of E1
and N5, N6 and N11 of E2) can also modulate HCVpp entry. Further studies will
be necessary to determine whether these mutations affect receptor binding or the
fusion properties of HCV envelope glycoproteins.

MECHANISMS OF HCV ENTRY

Virus attachment to receptors initiates a series of events that lead to virus entry. For
enveloped viruses, the entry process is controlled by viral surface glycoproteins
that undergo triggered conformational changes from a metastable state to a lower
energy state. This structural change leads to the exposure of a buried functional
element, named the fusion peptide and is believed to provide the energy required
for the merging of the lipid bilayers (reviewed in Colman and Lawrence, 2003).
So far, viral fusion proteins have been shown to fall into two different structural
classes designated as class I and II (reviewed in Earp et al., 2005). Class I fusion
proteins possess N-terminal or N-proximal fusion peptides, and they are synthesized
as a precursor that is cleaved into two subunits by host cell proteases. In some
cases (e.g., influenza HA), the two subunits remain associated through a disulfide
bond, whereas in others (e.g. HIV Env) the two subunits remain associated through
noncovalent interactions. The proteolytic processing event creates the metastable
state of the fusion protein (Colman and Lawrence, 2003). In their native metastable
conformation, class I fusion proteins form trimeric spikes at the surface of the virions
with the fusion subunit being highly helical. Upon a fusion trigger event (receptor
binding at the cell surface or low pH in endosomes), the trimeric proteins transiently
form an extended conformation allowing the hydrophobic fusion peptide to insert
into the target membrane. Protein refolding leads then to the formation of very stable
trimeric structures in which both the N-proximal fusion peptide and the C-proximal
membrane anchor are juxtaposed at the same end to allow virus and cell membrane
connection and hemifusion (reviewed in Colman and Lawrence, 2003).

Class II viral fusion proteins have a completely different structure. They are
predominantly non-helical, instead having a beta-sheet type structure; they are not
cleaved during biosynthesis; and they possess an internal fusion peptide with a loop
conformation (reviewed in Earp et al., 2005). The proteins are oriented parallel to
the membrane, and they have a three-domain architecture with domain I beginning
at the N-terminus, domain II containing the internal fusion loop, and domain III

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being at the C-terminus. In addition, class II fusion proteins are synthesized as

a complex with a second membrane glycoprotein (prM for flaviviruses; pE2 for
alphaviruses). Newly synthesized E and prM proteins of the tick borne encephalitis
virus associate to form noncovalent heterodimers (Fig. 4) that are incorporated into
immature virions by budding into the ER lumen (Allison et al., 1995; Mackenzie
and Westaway, 2001). These particles are then transported through the secretory
pathway and shortly before release from the cell, the activation of the fusogenic
potential occurs by the cleavage of the accessory protein prM by a cellular furin
protease in the trans-Golgi network (Stadler et al., 1997). After prM cleavage, the
E protein exists as a metastable homodimers at the virion surface. The ectodomains
of the dimers are orientated antiparallel to one another (Rey et al., 1995; Lescar
et al., 2001; Modis et al., 2003). The architecture of the alphavirus Semliki Forest
virus spike is similar to that of tick borne encephalitis virus E, but in this case, the
metastable oligomer is a heterodimer of the fusion protein E1 and the companion
protein E2 with an associated small protein E3 (Lescar et al., 2001). In addition,

Fig. 4. Comparison of flavivirus and hepacivirus envelope proteins. In the Flaviviridae family, class
II fusion proteins (depicted in light grey) have been described in the flaviviruses (E protein of tick
born encephalitis and dengue viruses). They are synthesized as a complex with a second membrane
glycoprotein (depicted in dark grey). Shortly before release from the cell, activation of the fusogenic
potential occurs by cleavage of the accessory protein (arrow). HCV envelope glycoproteins are
supposed to belong to the class II fusion proteins, but contrary to flaviviruses, HCV envelope proteins
are highly glycosylated and are not matured by a cellular endoprotease during their transport through
the secretory pathway.

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contrary to flaviviruses, alphaviruses have been shown to bud from the plasma
membrane.

Both alphaviruses and flaviviruses enter target cells by receptor-mediated

endocytosis. The receptor recognition function is carried by the fusion protein itself
for the flaviviruses (E) and by the companion protein (E2) for the alphaviruses.
Exposure to the acidic pH of the endosomes triggers a major conformational change
of the envelope involving dissociation of the native homodimer (for flaviviruses)
or heterodimer (for alphaviruses) and the irreversible formation of homotrimers of
the fusion proteins (Earp et al., 2005; Mukhopadhyay et al., 2005).

Based on its classification in the Flaviviridae family, HCV envelope has been
proposed to contain a class II fusion protein (Yagnik et al., 2000). As found in
the case of alphaviruses and flaviruses, HCVpp entry is pH dependent (Bartosch
et al., 2003c; Hsu et al., 2003). These observations indicate that HCV may enter
the cells through endocytosis. The cell surface receptor(s) recognized by HCV
should therefore traffic cell-bound virions to endosomal compartments. However,
characterization of the route of HCV entry needs further investigations. Contrary
to what is observed for other class II envelope proteins, there is no evidence that
HCV envelope glycoproteins are matured by a cellular endoprotease during their
transport through the secretory pathway (Op De Beeck et al., 2004). In addition,
HCV envelope glycoproteins are highly glycosylated, whereas other described class
II envelope proteins contain a very low number of glycans (Fig. 4). Interestingly,
some of the glycans present on HCV envelope glycoproteins seem to be involved
in controlling HCV entry (Goffard et al., 2005).

There remains some controversy on the identity of HCV fusion protein. It has been
proposed that E1 might be a good candidate because sequence analyses suggest that
it might contain a putative fusion peptide in its ectodomain (Flint et al., 1999b; Garry
and Dash, 2003). On the other hand, potential structural homology with other class
II fusion proteins suggests that E2 could be the fusion protein (Yagnik et al., 2000).
Mutagenesis studies in the putative fusion peptides of the envelope glycoproteins
associated with HCVpp as described for the flavivirus envelope protein E (Allison
et al., 2001), should be helpful for further characterization of HCV fusion protein.
In addition, a high-resolution structure of HCV envelope glycoproteins would also
help understanding the fusion mechanism of the virus.

INHIBITION OF HCV ENVELOPE GLYCOPROTEIN FUNCTIONS BY

NEUTRALIZING ANTIBODIES
Because they are exposed at the surface of the virion, the envelope proteins are
targets of neutralizing antibodies. These antibodies block a viral infection by
inhibiting virion binding or membrane fusion. Understanding the mechanisms of
neutralization needs therefore a good knowledge of the mechanism of entry. The

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role of neutralizing antibodies in HCV infection and disease progression remained

unclear for a long time, largely because of the lack of assays to measure and quantify
their activity. Previous experiments showed that serum from a chronically infected
patient could neutralize HCV infectivity in a chimpanzee model, giving evidence
for antibody-mediated neutralization of HCV (Farci et al., 1994). Neutralizing
antibodies could also be identified by their ability to prevent HCV replication in a
lymphoid cell line (Shimizu et al., 1994; Shimizu et al., 1996).

The recent development of HCVpp offers the possibility to study HCV neutralization
with defined sequences of HCV envelope glycoproteins, and the use of HCVpp in
neutralization studies has been validated (Bartosch et al., 2003a). As determined
with HCVpp, it seems that the majority of chronically infected patients have
cross-reactive neutralizing antibodies (Logvinoff et al., 2004; Meunier et al.,
2005). In contrast, neutralizing antibodies have not been detected in several cases
of acute resolving infection (Logvinoff et al., 2004; Meunier et al., 2005), and the
detection of neutralizing antibodies in acutely infected individuals did not seem
to be associated with viral clearance (Logvinoff et al., 2004). However, another
study has shown in some patients a progressive emergence of a relatively strong
neutralizing response in correlation with a decrease in viremia (Lavillette et al.,
2005a). Further investigations on a large number of acutely infected patients will
be necessary to determine the role of neutralizing antibodies in controlling HCV
infections. Interestingly, it has been observed that HCVpp infectivity is enhanced
by human sera, and this enhancement of infectivity can partly mask the presence of
neutralizing antibodies (Lavillette et al., 2005a; Meunier et al., 2005). In addition,
HDL have been identified as the component responsible for serum-mediated
enhancement of infectivity (Meunier et al., 2005; Voisset et al., 2005).

For a long time, the HVR1 sequence of E2 has been proposed to be a major target
for neutralizing antibodies (Kato et al., 1993; Farci et al., 1996). However, data
obtained with the HCVpp model indicate that neutralizing epitopes located outside
of HVR1 also exist (Bartosch et al., 2003a). Interestingly, characterization of HCVpp
with monoclonal antibodies has allowed to identify conformation-dependent and
-independent neutralizing epitopes outside of HVR1 (Fig. 2)(Bartosch et al., 2003b;
Hsu et al., 2003; Keck et al., 2004; Op De Beeck et al., 2004). Conformation-
dependent human monoclonal antibodies have also allowed to identify three
immunogenic domains in E2 with neutralizing antibodies being restricted to
two of these domains (Keck et al., 2004). Whether E2 domains identified with
these monoclonal antibodies are similar to the antigenic structural and functional
domains of the envelope protein E of the flaviviruses (Rey et al., 1995) remains
to be determined.

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CONCLUSION
Studies of the biogenesis of HCV envelope glycoproteins have shown the pivotal
role of the transmembrane domains in the assembly of a noncovalent E1E2
heterodimer in the ER. More recently, the development of the HCVpp model has
allowed to investigate the role of E1E2 heterodimer in virus entry. Functional regions
in HCV envelope glycoproteins can now be identified and potential receptors can
also be validated. Entry is an essential step in the life cycle of a virus, which can
potentially be blocked by neutralizing antibodies or antiviral drugs that target the
envelope proteins of the virus. Understanding the viral and cellular components
involved in HCV invasion into the host cell, combined with a comprehension of
the mechanisms that govern this process, should therefore open the possibility of
developing new therapeutic approaches.

FUTURE TRENDS
The development of the HCVpp model has allowed to initiate the characterization of
the entry function of HCV envelope glycoproteins. The use of HCVpp will continue
to provide additional information on the role of HCV envelope glycoproteins in
viral entry. However, the recent development of a full-length clone that is infectious
in cell culture (see chapter 16) provides new opportunities to study the functions
of HCV envelope glycoproteins. A comparison of the properties of HCV envelope
glycoproteins produced in HCVpp and in this infectious clone will be very useful to
validate the data that have been generated during the past three years. In addition,
this infectious clone will allow for the first time to decipher the role of HCV
envelope glycoproteins in virion assembly. Finally, obtaining a high-resolution
structure of HCV envelope glycoproteins will also be necessary to understand the
fusion mechanism of this virus.

ACKNOWLEDGMENTS
We thank Sophana Ung for preparing the illustrations. Our research was supported
by EU grant QLRT-2000-01120 and QLRT-2001-01329 and grants from the "Agence
Nationale de Recherche sur le Sida et les hépatites virales" (ANRS), INSERM "ATC-
Hépatite C" and the "Association pour la Recherche sur le Cancer" (ARC).

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Chapter 5

HCV NS2/3 Protease

Sarah Welbourn and Arnim Pause

ABSTRACT
The hepatitis C virus NS2/3 protein is a highly hydrophobic protease responsible
for the cleavage of the viral polypeptide between non-structural proteins NS2 and
NS3. However, many aspects of the NS2/3 protease's role in the viral life cycle and
mechanism of action remain unknown or controversial. NS2/3 has been proposed
to function as either a cysteine or metalloprotease despite its lack of sequence
homology to proteases of known function. In addition, although shown to be required
for persistent infection in a chimpanzee, the role of NS2/3 cleavage in the viral
life cycle has not yet been fully investigated due to the lack of an in vitro system
in which to study all aspects of HCV replication. However, several recent studies
are beginning to clarify possible roles of the cleaved NS2 protein in modulation
of host cell gene expression and apoptosis.

INTRODUCTION
The NS2/3 protease is the first of two virally encoded proteases required for HCV
polyprotein processing. Extending from amino acids 810-1206, NS2/3 is the first
non-structural (NS) protein translated and is responsible for the intramolecular
cleavage between NS2 and NS3 (see Fig. 1). The amino terminus of NS2 is cleaved
from the adjacent p7 protein by host signal peptidases in a membrane-dependent

C E1 E2 P7 NS2 NS3 NS4A NS4B NS5A NS5B

810 NS2/3 1206

NS2 NS3
810 1026 1027 1206

Fig. 1. The HCV NS2/3 protease. The NS2/3 protease is shown in the context of the HCV polyprotein.
NS2 and the protease domain of NS3 (from aa 810 to 1206) constitute NS2/3, which undergoes
autocatalytic cleavage between aa 1026 and 1027.

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manner, while the chymotrypsin-like serine protease located in NS3 is responsible

for the cleavage at the NS3/4A and downstream junctions. The HCV NS2/3 protein
is an autoprotease whose activity is separate from NS3 protease functions (see
Chapter 6). Although many studies have focused on the residues and sequences
required for efficient NS2/3 processing, the exact nature of the protease has still
not been firmly established, with it being proposed to function as either a novel
cysteine or metalloprotease. Furthermore, although all NS proteins are proposed to
play a role in viral replication, the exact functions of HCV NS2/3, as well as cleaved
NS2 remain largely unexplored; however, some interesting potential functions
have emerged in recent years. This chapter will focus on the known properties of
the NS2/3 protease as well as the possible functions of both the NS2/3 protease
and the NS2 protein.

NS2/3 CATALYTIC CLEAVAGE

GENERAL STRUCTURAL FEATURES OF NS2/3
The NS2/3 protease is responsible for the intramolecular cleavage of NS2 from
NS3 between aa 1026 and 1027 (Grakoui et al., 1993; Hijikata et al., 1993a). Fig.
2 shows the main structural and functional domains of the protein. NS2 contains a
highly hydrophobic N-terminal region suggested to contain multiple transmembrane
segments; however, this region is not required for efficient cleavage at the NS2/3
site (Hijikata et al., 1993a; Pallaoro et al., 2001; Thibeault et al., 2001). The minimal
domain for activity of the enzyme has been mapped to aa 907-1206 (Pallaoro et al.,
2001). This encompasses the C-terminal portion of NS2, immediately following the
hydrophobic region, as well as the N-terminal protease domain of NS3. Although
these sequences are required and sufficient for cleavage activity, processing is
not dependent the NS3 serine protease activity (Grakoui et al., 1993; Hijikata et
al., 1993a). This differs from the NS2B protein of flaviviruses, in which the NS3

NS3 Structural Zinc

Binding Sites
Hydrophobic C1123 C1125C1171 H1175
Region H952 E972 C993
* * * ** **

810 907 1026 1206

Minimal Region for NS2/3 activity

NS2 NS3

Fig. 2. Functional domains of the NS2/3 protease. The NS2/3 protease encompasses an N-terminal
hydrophobic region, with a minimal domain required for activity between aa 907 and 1206. Residues
in NS2 required for NS2/3 processing (H942, E972, C993), as well as residues in NS3 responsible
for the coordination of a zinc atom are shown.

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protease performs a cis-cleavage at the NS2B site and then uses NS2B as a co-
factor for the processing of the downstream NS polypeptide (Chambers et al., 1991;
Chambers et al., 1990; Falgout et al., 1991).

NS2/3 PROCESSING REQUIREMENTS

The HCV NS2/3 protease shows no sequence motifs typical of known proteases;
however, sequence alignments show similarity with the GBV NS2/3 protein as well
as the bovine viral diarrhea virus (BVDV) NS2/3 protein (Lackner et al., 2004).
Residues H952, E972 and C993 are conserved among all genotypes of HCV and
mutation of H952 or C993 to alanine completely inhibits NS2/3 cleavage activity
while a glutamic acid 972 to glutamine substitution also significantly affects
processing (Grakoui et al., 1993; Hijikata et al., 1993a). Furthermore, although
NS3 serine protease activity is not required for NS2/3 processing, the full NS3
protease domain must be present and cannot be substituted for another NS protein
(Santolini et al., 1995). In addition, mutation of cysteine residues 1123, 1127 and
1171 in NS3, which together with H1175 participate in the coordination of a zinc
molecule (Kim et al., 1996; Love et al., 1996), abolishes both NS3 and NS2/3
activities (Hijikata et al., 1993a), presumably by disrupting folding of the enzymes.
This therefore suggests that the NS3 protease domain is required to play a structural
role in the folding of the enzyme.

Proper folding of the NS2/3 protein and cleavage site plays an important role
in the efficiency of NS2/3 processing. Residues surrounding the cleavage site,
WRLL↓APIT, are highly conserved between HCV genotypes but are remarkably
resistant to mutations (Hirowatari et al., 1993; Reed et al., 1995). Only mutations
severely affecting the conformation of the cleavage site (such as deletion or proline
substitution of P1 or P1') severely inhibit cleavage. Furthermore, NS4A-derived
peptides that upon binding cause a conformational rearrangement of the NS3 N-
terminus are potent inhibitors of NS2/3 activity, likely by altering the positioning
of the cleavage site (Darke et al., 1999; Thibeault et al., 2001). The presence of
microsomal membranes or non-ionic detergents has been found to be required for in
vitro processing at the NS2/3 site in certain genotypes (Pieroni et al., 1997; Santolini
et al., 1995), while increasing the efficiency of cleavage of others (Grakoui et al.,
1993; Santolini et al., 1995), suggesting the hydrophobic environment is necessary
for proper folding of the enzyme and positioning of the cleavage site. Similarly,
Waxman et al. (2001) have demonstrated the requirement for the ATP hydrolyzing
ability of molecular chaperone HSP90 for efficient cleavage in in vitro and cell
based assays. A similar phenomenon has been described for the BVDV NS2/3
protein where a cellular DnaJ chaperone protein, Jiv, has been found to associate
with and modulate NS2/3 activity, possibly by causing a conformational change
in the protein (Rinck et al., 2001). Although the mechanisms are still unclear, this
could point to a role of cellular chaperones in inducing/maintaining the proper
conformation of NS2/3 required for cleavage.

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MECHANISM OF ACTION: CYSTEINE OR METALLOPROTEASE?

Initial studies showing NS2/3 activity is inhibited by EDTA and stimulated by zinc
led to the early suggestion that NS2/3 functions as a zinc-dependent metalloprotease
(Hijikata et al., 1993a). However, with the discovery of the importance of zinc for
the structural integrity of the NS3 protease domain, others have proposed NS2/3 may
be a novel cysteine protease with a catalytic dyad comprised of H952 and C993 with
the possible involvement of E972 as the third residue of a catalytic triad. Inhibition
studies both in in vitro translation systems and with purified proteins have failed to
yield a definite classification (Pallaoro et al., 2001; Pieroni et al., 1997; Thibeault et
al., 2001). Although inhibited by metal chelators such as phenanthroline and EDTA,
this inhibition is relieved by the addition of ZnCl2, CdCl2 or MgCl2. This could
therefore point to a structural rather than catalytic role for the zinc molecule as Cd
has not traditionally been able to functionally replace Zn in other metalloproteases
(Angleton and Van Wart, 1988; Cha et al., 1996; Holland et al., 1995). However,
although classical cysteine protease inhibitors iodoacetamide and N-ethylmaleimide
show strong inhibition of NS2/3 processing, no single cysteine has been found to
be more susceptible to these alkylating agents (Pallaoro et al., 2001).

Recently, conserved His, Cys and Glu have also been found to be present in BVDV
strains and required for NS2/3 cleavage in vitro (Lackner et al., 2004), suggesting
a similar mechanism of action of the two proteases. However, several differences
exist. In addition to the necessity of the N-terminal hydrophobic region of NS2,
BVDV NS2/3 does not require the full NS3 protease domain for activity, but rather
possesses a conserved zinc-binding site within NS2 itself (Lackner et al., 2004).
Although no traditional metal-binding sequences have been identified in HCV NS2,
the presence of an additional catalytic zinc in NS2 or a catalytic role for the NS3
zinc molecule cannot be definitely ruled out. The elucidation of the so far unknown
crystal structure of NS2/3 should bring important insights into the mechanism of
cleavage of this enzyme.

NS2/3 BIMOLECULAR CLEAVAGE

Bimolecular cleavage of NS2/3 has been shown to occur, albeit inefficiently, in cell
transfection experiments (Grakoui et al., 1993; Reed et al., 1995). In this system,
NS2/3 proteins with mutations/deletions in either the NS2 or NS3 domains could
support cleavage provided the missing functional region was co-expressed on a
separate polypeptide. In addition, catalytically inactive NS2/3 mutants were also
found to inhibit processing of a wild-type protein when expressed in trans. The
observation that a recombinant NS2/3 protein forms dimers in vitro is consistent
with these findings (Pallaoro et al., 2001). However, no trans cleavage has been
observed using purified proteins (Pallaoro et al., 2001; Thibeault et al., 2001).
Interestingly, NS2/3 activity in vitro was found to be concentration dependent,
supporting the notion that dimer formation is essential for the reaction (Pallaoro et
al., 2001). Dimitrova et al. (2003) have also demonstrated the homo-association of

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the NS2 protein in various systems and suggest that the cleavage between NS2 and
NS3 could potentially be performed by dimers of NS2/3 encoded on neighbouring
polyprotein chains. As NS2/3 cleavage is widely believed to be an intramolecular
event, the significance of bimolecular cleavage in the polyprotein processing events
of HCV infection in vivo remains to be determined.

ROLE OF NS2/3 CLEAVAGE IN VIRAL REPLICATION

The role of the NS2/3 protease in HCV replication remains to be fully understood.
NS2/3 cleavage is required for viral replication in vivo, as demonstrated by an
HCV clone devoid of NS2/3 activity that fails to cause a persistent infection in a
chimpanzee (Kolykhalov et al., 2000). However, NS3-3' UTR subgenomic replicons
not encoding the NS2 protein replicate efficiently in Huh-7 cells (Lohmann et al.,
1999), suggesting NS2/3 is not strictly required for genome replication.

If cleavage at the NS2/3 site occurs solely for the release of the NS2 protein, what
is the advantage for the virus of encoding two distinct proteases for polyprotein
processing? Although several roles have been proposed for the cleaved NS2 protein,
the NS2/3 protease itself appears unique in that its activity subsequently causes
its inactivation. However, potential regulation of the cleavage reaction could have
other implications for the viral life cycle, as is known for BVDV NS2/3 processing.
BVDV stains are present in two forms, non-cytopathic (noncp) which expresses
primarily uncleaved NS2/3 and has the ability to cause persistent infection and
cytopathic (cp) strains expressing cleaved NS3 (Donis and Dubovi, 1987; Pocock et
al., 1987). For this pestivirus, RNA replication levels have been shown to correlate
with amount of cleaved NS3 protein (Lackner et al., 2004), whereas the uncleaved
NS2/3 is required for viral infectivity (Agapov et al., 2004). Evolution of a cp
strain from a non-cp strain occurs through the activation of the NS2/3 cleavage
by a variety of mutations, deletions, duplications and rearrangements within the
NS2 region (Kummerer et al., 1998; Meyers et al., 1992; Tautz et al., 1996; Tautz
et al., 1994). However, it has recently been suggested that BVDV NS2/3 is an
autoprotease whose temporal regulation is involved in modulating the different
stages of RNA replication and viral morphogenesis (Lackner et al., 2004). Whether
HCV NS2/3 could perform a similar regulatory role remains to be determined.
Although NS2/3 processing appears to be a very efficient event in cell expression
systems, the possible role for an uncleaved NS2/3 precursor in the complete viral
life cycle has not been ruled out.
.
NS2 AS PART OF THE REPLICATION COMPLEX?
HCV RNA replication has been proposed to occur via the formation of a
membrane bound replication complex that comprises the association of the NS
proteins required for genome replication (NS3-5B). However, due to the lack of
an efficient cell culture system to study the viral life cycle, studies focusing on the

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replication complex have been so far limited to the subgenomic replicon system
(see Chapter 11), where NS2 is not expressed. Several studies have indicated that
NS2 is an integral membrane protein that is targeted to the endoplasmic reticulum
(ER) (Santolini et al., 1995; Yamaga and Ou, 2002). Interestingly, NS2 has been
found by one group to be inserted into the membrane only when expressed in the
context of the NS2/3 protein, and only after cleavage from NS3 (Santolini et al.,
1995). NS2 has also been found to interact with all other HCV NS proteins in in
vitro pull-down, as well as cell-based co-localization and co-immunoprecipitation
experiments (Dimitrova et al., 2003; Hijikata et al., 1993b). Therefore, although
not required for RNA replication, the possible presence of NS2 in this complex as
an accessory protein is plausible and warrants further investigation.

ROLES OF CLEAVED NS2

HCV NS2 IS AN INTEGRAL MEMBRANE PROTEIN

The NS2 protein derived from the cleavage of NS2/3 is inserted into the ER
membrane through its N-terminal hydrophobic domain. However, the exact
mechanisms of translocation as well as the membrane topology of the protein
remain controversial. Membrane association has been found to be dependent on
SRP-SRP receptor targeting (Santolini et al., 1995). It was originally proposed that
a signal sequence present in upstream p7 was required for membrane association
co-translationally, although NS2 translocation has subsequently been demonstrated
by several groups to be p7 independent (Santolini et al., 1995; Yamaga and Ou,
2002). Furthermore, although the cleavage at the p7-NS2 junction is performed in
a membrane-dependent fashion by signal peptidase (Lin et al., 1994; Mizushima
et al., 1994) and the presence of membranes is stimulatory (and for some strains
required) for NS2/3 cleavage, one group has shown that the integration of NS2 into
the membrane is performed post-translationally, and only after cleavage from NS3
(Santolini et al., 1995). However, Yamaga and Ou (2002) have since proposed that
translocation could occur co-translationally and therefore the exact mechanisms of
integration remain unclear. The amino terminal region of NS2 is likely to span the
membrane several times (Pallaoro et al., 2001; Yamaga and Ou, 2002). However,
the exact number of transmembrane domains, as well as the orientation of the
protein in the membrane have not been conclusively determined.

NS2 AND NS5A HYPERPHOSPHORYLATION

HCV NS5A has many roles in both RNA replication and the modulation of the host
cell environment during infection and has been found to be present in two distinct
phosphorylated forms: p56 and p58 (see Chapter 9). Liu et al. have reported the
importance of NS2 for the generation of hyperphosphorylated NS5A (p58) (Liu
et al., 1999). Using plasmids expressing various sections of the HCV polyprotein
in transient transfection experiments, they demonstrate the requirement of NS2
generated by the cleavage of NS2/3 for the formation of p58. However, while

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performing similar experiments, other groups have demonstrated the appearance

of p58 without the presence of NS2 (Koch and Bartenschlager, 1999; Neddermann
et al., 1999). Indeed, Neddermann et al. (1999) therefore suggested that NS2
itself is not required for the hyperphosphorylation process, but rather that it could
be the authentic N-terminus of NS3, generated by NS2/3 cleavage, that is of
importance.

NS2 INHIBITION OF GENE EXPRESSION

NS2 may also play a role in modulating cellular gene expression in infected cells.
One study by Dumoulin et al. (2003) found that NS2 exerted a general inhibitory
effect on the expression of a reporter gene expressed from a variety of different
promoters (human ferrochelatase promoter, NFkappaB binding sites, SV40
promoter/enhancer sequences, full length, as well as minimal TNF-alpha promoters
and cytomegalovirus immediate-early promoter) in several different hepatic and
non-hepatic cell types. The amino-terminal (810-940) region of NS2 was sufficient
to cause this effect, suggesting inhibition of gene expression is not dependent on
the activity of the NS2/3 protease itself. It was therefore suggested that NS2 could
potentially regulate host cell protein levels by interfering with a general aspect of
transcription or translation. Indeed, several other HCV-encoded proteins, including
core (Chapter 3), NS4B (Chapter 8) and NS5A (Chapter 9), have been demonstrated
to alter cellular gene expression though a variety of mechanisms (Kato et al., 1997;
Kato et al., 2000; Naganuma et al., 2000; Ray et al., 1995). This aspect of NS2
function will require further confirmation and careful investigation as it indicates
a potential role for NS2 in the modulation of the host cell environment which has
important implications for both the establishment of persistent infection and the
pathogenesis of chronic HCV.

NS2 AND APOPTOSIS

In order to establish a persistent infection, many viruses have evolved mechanisms
to interfere with cellular apoptosis. In this manner, the virus is then able to replicate
to sufficient levels without the elimination of the host cell. Several HCV proteins
have been implicated in the modulation of cell signalling and apoptosis, including
core, E2, NS5A and NS2 (Gale et al., 1997; Honda et al., 2000; Machida et al.,
2001; Ruggieri et al., 1997). Machida et al. (2001) have reported that Fas-mediated
apoptosis is inhibited in transgenic mice expressing HCV core, E1, E2 and NS2
proteins. The expression of these proteins in the liver prevented cytochrome c release
from the mitochondria as well as preventing the activation of caspase 9 and caspase
3/7, but did not affect caspase 8. Therefore, this implicates these HCV proteins in the
mitochondrial intrinsic apoptotic pathway, which involves mitochondrial membrane
permeabilization and the release of pro-apoptotic factors, resulting in cell death.
Furthermore, Erdtmann et al. (2003) showed that NS2 inhibits CIDE-B-induced
apoptosis in co-expression experiments. CIDE-B (cell death-inducing DFF45-
like effector) is a mitochondrial pro-apoptotic protein whose overexpression has

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been shown to induce cell death (Inohara et al., 1998). CIDE-B-induced apoptosis
requires mitochondrial localization and dimerization of the protein, both of which
are mediated by a region in its C-terminal domain (Chen et al., 2000). NS2 was
found to interact specifically with the C-terminal region of CIDE-B and block
cytochrome c release from the mitochondria as well as cell death (Erdtmann et
al., 2003). NS2 could therefore potentially prevent the dimerization of CIDE-B
required for activity. However, the mechanism of inhibition remains unclear as NS2
is thought to be localized at the ER membrane. In this case, NS2 could potentially
bind and sequester CIDE-B, preventing its localization at the mitochondria.

The roles of mature cleaved NS2 remain largely unexplored. Although some possible
functions have been proposed and are described here, the lack of an efficient cell
culture system remains a major hurdle in identifying the main tasks of NS2 in the
various events of the viral life cycle. Furthermore, it has been observed that NS2
is a short-lived protein in replicon cells (Franck et al., 2005). Franck et al. (2005)
showed that NS2 is a target for phosphorylation by CK2 and is subsequently rapidly
degraded by the proteasome. This appears to be a ubiquitin-independent process and
the exact mechanisms involved have yet to be identified. However, the regulation of
this process could have important implications for the understanding of the various
functions of NS2 and the sequential events of the viral life cycle.

CONCLUSIONS
Much work is still required in the study of the NS2/3 protease. Although several
studies over the past decade have focussed on NS2/3 cleavage, the catalytic
mechanism of the enzyme remains controversial. Initial attempts at characterizing
the enzyme were limited to in vitro and cell expression systems and despite the
development in recent years of in vitro systems in which the processing reaction can
be studied using purified recombinant proteins, a definitive classification has not yet
been determined. A three dimensional structure of NS2/3 is very much needed and
will likely yield important insights into the mechanism of action of the enzyme.

Similarly, a robust cell culture system for the study of the viral life cycle is of
urgent need (see Chapter 16). Such a system will be crucial to precisely define the
roles of NS2/3 cleavage and the NS2 protein in the complete viral life cycle. Of
particular interest are the observations that NS2 could potentially modulate the host
cell environment during HCV infection through interference with gene expression
and cellular apoptosis. However, it will be necessary to validate these findings in
a more physiologically relevant setting.

Although its mode of action is unclear, NS2/3 cleavage is absolutely required for
persistent viral infection in a chimpanzee. The HCV NS2/3 protease shares no
obvious sequence homology to any known proteases in the animal kingdom and

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would therefore make an attractive target for antiviral therapy. The elucidation
of the crystal structure of NS2/3, its mechanism of action and precise functions
in replication will help to generate important information for the development of
strategies for inhibition of NS2/3 processing, which could become the basis for
novel HCV therapies in the future.

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Chapter 6

HCV NS3-4A Serine Protease

Chao Lin

ABSTRACT
The 9.6 kb plus-strand RNA genome of HCV encodes a long polyprotein precursor
of ~3,000 amino acids, which is processed by cellular and viral proteases to 10
individual proteins. One of the HCV proteases, NS3-4A serine protease, is a non-
covalent heterodimer consisting of a catalytic subunit (the N-terminal one-third
of NS3 protein) and an activating cofactor (NS4A protein), and is responsible for
cleavage at four sites of the HCV polyprotein. HCV NS3-4A protease is essential
for viral replication in cell culture and in chimpanzees, and has been considered as
one of the most attractive targets for developing novel anti-HCV therapies. However,
discovery of small-molecule, selective inhibitors against HCV NS3-4A protease as
oral drug candidates has been hampered by its shallow substrate-binding groove and
the lack of robust, reproducible viral replication models in cell culture or in small
animals. Nevertheless, decade-long intense efforts by many groups have largely
overcome these two obstacles and provided fruitful understanding of its biological
functions, biochemistry, and three-dimensional structures, culminating in recent
demonstration of proof-of-concept anti-HCV activities in patients. This chapter
will review key findings in these areas, and focus on the discovery and clinical
development of HCV NS3-4A protease inhibitors as novel antiviral therapies.

INTRODUCTION
The hepatitis C virus (HCV) epidemic, affecting ~170 million people worldwide,
has been widely discussed (Memon and Memon, 2002; Wasley and Alter, 2000). The
current standard therapy for chronic hepatitis C patients is a combination of weekly
injections of pegylated interferon (IFN)-α, and daily oral doses of ribavirin (for a
review, see Anonymous, 2002; Strader et al., 2004 and references therein). Both
drugs are indirect antivirals because they do not target a specific HCV protein or
RNA element. A sustained viral response (SVR), which is defined as treated patients
remaining HCV-free (undetectable viral load) for 6 months after the termination of
therapy, is achieved in only half of the treated patients and in less than half of patients
with genotype 1 HCV or with high viral load (Fried et al., 2002; Hadziyannis et
al., 2004; Manns et al., 2001). The standard therapy is associated with considerable
adverse effects, including depression, fatigue, and "flu-like" symptoms caused by
IFN-α, and hemolytic anemia by ribavirin. There is a huge unmet medical need

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for orally available, small-molecule, direct anti-HCV drugs to provide hepatitis C

patients more effective treatments with fewer side effects.

HCV, a member of the Flaviviridae family of viruses, has a 9.6 kb plus-strand RNA
genome that encodes a long polyprotein precursor of ~3,000 amino acids, which
is processed proteolytically upon translation by both cellular and viral proteases
to at least 10 individual proteins, including four structural proteins (C, E1, E2 and
p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and
NS5B) (Fig. 1) (for a review see Lindenbach and Rice, 2001). The NS3 protein is
a multi-functional protein, with a serine protease domain in its N-terminal one-third
and a helicase domain in the C-terminal two-third (reviewed in chapter 7). The
NS3-4A serine protease is a non-covalent, heterodimer complex formed by two
HCV-encoded proteins, the N-terminal serine protease domain of NS3 (catalytic
subunit) and the NS4A cofactor (activation subunit). The NS3-4A serine protease
is responsible for the proteolytic cleavage at four junctions of the HCV polyprotein
precursor: NS3/NS4A (self cleavage), NS4A/NS4B, NS4B/NS5A, and NS5A/
NS5B (Fig. 1) (Bartenschlager et al., 1993; Bartenschlager et al., 1995b; Failla et
al., 1995; Grakoui et al., 1993a; Grakoui et al., 1993b; Hijikata et al., 1993b; Kim
et al., 1996; Lin and Rice, 1995; Lin et al., 1995; Tanji et al., 1995; Tomei et al.,
1993). HCV encodes four viral enzymes in its nonstructural protein region: NS2-3
autoprotease (reviewed in chapter 5) and NS3-4A serine protease (reviewed in this
chapter), NS3 helicase (reviewed in chapter 7) and NS5B RNA-depdendent RNA
polymerase (reviewed in chapter 10), all of which are essential for HCV replication
or infectivity in chimpanzees (Kolykhalov et al., 2000). Among them, NS3-4A serine
protease and NS5B RNA-dependent RNA polymerase are generally considered to
be the most attractive targets for design of new anti-HCV oral drugs.

The success of HIV protease inhibitor drugs demonstrates that viral proteases,
such as the HCV NS3-4A protease, could be excellent targets for a structure-based
drug design approach. However, the shallow substrate-binding groove of the HCV

Fig. 1. A schematic diagram of the HCV genome. The 5' and 3' untranslated regions (UTR) are shown
with putative secondary structures. The polyprotein encoded by the long open reading frame is shown
as a long box, in which individual mature protein products are labeled as core (C), envelope proteins
1 and 2 (E1 and E2), p7, followed by six nonstructural proteins (NS) 2, 3, 4A, 4B, 5A, and 5B. The
cleavage sites are marked for cellular signal peptidase (filled triangle), HCV NS2-3 auto-protease
(filled arrow), and NS3-4A serine protease (open arrow).

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NS3-4A serine protease observed in an X-ray crystal structure (Kim et al., 1996)
suggested that discovery of a potent, small-molecule, and orally available drug
candidate would be an enormously challenging task. Despite of the lack of a robust
and consistent HCV infection cell culture, a subgenomic replicon system developed
by Lohmann et al. (1999) became the workhorse as the standard assay of antiviral
activity of the HCV NS3-4A protease inhibitors. In addition, the lack of a robust
HCV infection model in small animals has generally forced scientists to rely on
a combination of anti-HCV activity in cell culture and animal pharmacokinetics
as surrogate indicators of efficacy prior to clinical trials in human. Nevertheless,
significant progress has been made in recent years to identify potent small-molecule
inhibitors against the HCV protease. Clinical proof-of-concept for HCV NS3-4A
protease inhibitors has recently been obtained with BILN 2061 (a non-covalent
inhibitor) and VX-950 (a covalent but reversible inhibitor). Viral load in chronic
hepatitis C patients was reduced by 2-3 log10 after a treatment with BILN 2061
(Lamarre et al., 2003) or VX-950 (Reesink et al., 2005) for 2–3 days. At the end
of a 14-day treatment with VX-950, up to a 4-log10 reduction in HCV viral load
was observed, while in some patients the virus became undetectable (<10 IU/mL)
by day 14 (Reesink et al., 2005).

BIOLOGICAL FUNCTIONS

THE PRESENCE OF A SERINE PROTEASE IN HCV NS3 PROTEIN

In 1989, two groups presented seminal comparative sequence studies suggesting
the presence of a trypsin/chymotrypsin-like serine protease in the N-terminal one-
third of the NS3 protein of flaviviruses and pestiviruses (Bazan and Fletterick,
1989; Gorbalenya et al., 1989a). Although the identity of the HCV proteins had not
been determined yet at that time, these groups showed that HCV might encode a
homologous trypsin/chymotrypsin-like serine protease as well. A catalytic triad of
His1083, Asp1107, and Ser1165 (based on the polyprotein number of HCV-H strain)
(Fig. 2A) was identified by sequence alignment of the HCV NS3 protein with many
known viral and cellular serine proteases, which belong to the trypsin/chymotrypsin
superfamily of serine proteases (Bazan and Fletterick, 1989; Bazan and Fletterick,
1990; Gorbalenya et al., 1989a; Miller and Purcell, 1990). In this chapter, a
numerical system starting with the N terminus of the HCV NS3 protein itself will be
used, in which the number of this catalytic triad would be His57, Asp81, and Ser139.
Mutagenesis studies of the catalytic triad by several laboratories showed that the
HCV NS3 serine protease is necessary for cleavage at four junctions in the HCV
polyprotein, including NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B
sites (Bartenschlager et al., 1993; Eckart et al., 1993; Grakoui et al., 1993a; Hijikata
et al., 1993a; Manabe et al., 1994; Tomei et al., 1993). Substitution of any of the
catalytic triad residues, His57, Asp81, or Ser139, abolished the cleavage at these four
junctions, but have no effect on cleavage at other sites of the HCV polyprotein

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Fig. 2. Sequence alignment of the HCV NS3-4A serine protease and its substrates. The genotypes (1a,
1b, 2a, 2b, and 3a) are indicated at the left, and the strain names are shown in a bracket.

(A) HCV NS3 serine protease domain. The catalytic triad, His57, Asp81, and Ser139 are labeled with
a filled triangle, the zinc-binding residues (Cys97, Cys99, Cys145, and His149) with an open triangle,
the S1 pocket residues (Leu135, Phe154, and Ala157) with a filled arrow, and the in vitro resistance
mutations (Arg155, Ala156, and Asp168) with an open arrow.

(B) HCV NS4A cofactor. The central region of NS4A that is required for activation of the NS3 serine
protease is indicated with an open box. The key hydrophobic residues of NS4A that interacts with
NS3 are highlighted with an underscore.

(C) The substrate sequences for the HCV NS3-4A serine protease. The scissile peptide bond is indicated
by a solid arrow. A total of 10 amino acids are shown for both the P- and the P'-sides. Residues that
are conserved among different cleavage sites are highlighted with an underscore.

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(C/E1, E1/E2, E2/p7, p7/NS2, and NS2/NS3), all of which are located upstream
of the four NS3 serine protease-dependent cleavage sites (Fig. 1). Cleavage at
the NS2/NS3 junction requires the presence of both NS2 and the N-terminal 180
residues of NS3, i.e., the serine protease domain, but not the catalytic triad of the
serine protease per se. The NS2-3 protease will be the topic of another chapter in
this book (reviewed in chapter 5).

THE MINIMAL DOMAIN OF SERINE PROTEASE: N-TERMINAL 180 AMINO

ACIDS
The 631-residue HCV NS3 protein is a dual-function protein, containing the
trypsin/chymotrypsin-like serine protease in the N-terminal region and a helicase
in the C-terminal region (Gorbalenya and Koonin, 1993; Gorbalenya et al.,
1989b). Co-transfection studies using constructs in which either the catalytic
triad of the serine protease was mutated or the entire protease domain was deleted
demonstrated that the NS3 serine protease domain, in the absence of its C-terminal
helicase counterpart, is capable of mediating cleavage of polyprotein substrates
(Bartenschlager et al., 1994; Lin et al., 1994; Tanji et al., 1994b). The minimal
sequences required for a functional serine protease activity were determined by
these groups to be the N-terminal 180 amino acids of the NS3 protein. Deletion of

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up to 14 residues from the N terminus of the NS3 protein is tolerated, although a

further deletion of the N-terminal 22 amino acids resulted in significantly poorer
processing of HCV polyprotein. On the other hand, deletions from C terminus
of this minimal serine protease domain completely abolished proteolytic activity
(Bartenschlager et al., 1994; Failla et al., 1995; Tanji et al., 1994b).

THE HCV NS3 SERINE PROTEASE-MEDIATED CLEAVAGE OF HCV

POLYPROTEIN
The N-terminal amino acid sequences of the HCV NS3 serine protease-dependent
cleavage products, including NS4A, NS4B, NS5A, and NS5B were determined by
radioactive labeling of the HCV polyprotein with specific amino acids followed
by N-terminal sequencing (Grakoui et al., 1993a). The nomenclature of Schechter
and Berger (Schechter and Berger, 1967), which has been widely accepted for
description of the proteases and the corresponding substrates, will be used for
the HCV NS3-4A serine protease, its substrates and inhibitors in this review. A
decapeptide substrate for the HCV NS3-4A protease, with 6 residues on the N-
terminal side and 4 residues on the C-terminal side (Fig. 2C and 4A), would be
described as NH2-P6-P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-OH, with the scissile bond
locating between the P1 and P1' residues. The cleavage products of this substrate
would be the P-side product (NH2-P6-P5-P4-P3-P2-P1-OH) and the P'-side product
(NH2-P1'-P2'-P3'-P4'-OH). The pockets on the HCV NS3-4A protease in which
the P1 side chain binds would be called the S1 pocket, and so forth for S2, S3, S4,
S5, or S6 pockets and S1', S2', S3', or S4' pockets. Sequence alignment of these
cleavage sites among various HCV isolates indicates that a consensus sequence
would include the following elements: an acidic residue (Asp or Glu) at the P6
position, a thiol-terminating residue (Ser for the NS3/NS4A and Thr for the other
three sites) at the P1 position, and a small side chain residue (Ala or Ser) at the P1'
position (Fig. 2C).

Site-directed mutagenesis studies showed that blockage of any one of these four
junctions has little or no impact on cleavage at other sites (Kolykhalov et al.,
1994; Leinbach et al., 1994), which is consistent with kinetic analyses that have
demonstrated a preferential, but clearly not obligatory, order of cleavage of the
NS polyprotein by the NS3-4A serine protease (Bartenschlager et al., 1994; Lin et
al., 1994; Tanji et al., 1994a). However, proteolysis of the NS3/NS4A junction is
believed to be a co-translational, cis-cleavage event since an NS3-NS4A precursor
were not detected and this cleavage was insensitive to dilution (Bartenschlager et al.,
1994; Lin et al., 1994; Tanji et al., 1994a). The other three junctions can be cleaved
by the NS3-4A serine protease in trans (Bartenschlager et al., 1994; Failla et al.,
1994; Lin et al., 1994; Tanji et al., 1994a; Tanji et al., 1994b). However, it is possible
that all these proteolysis events occur in a localized, i.e. cis-cleavage environment,
since the NS3-4A protease is expressed as part of the same polyprotein molecule
as all of its substrates. In trans-cleavage reactions, processing at the NS5A/NS5B

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site occurred more rapidly than those at the NS4A/NS4B and NS4B/NS5A sites
since rather stable NS4A-NS4B-NS5A processing intermediates were detected
(Bartenschlager et al., 1994; Failla et al., 1995; Lin et al., 1994). Two additional
cleavage sites in NS4B and NS5A have been identified (Kolykhalov et al., 1994;
Markland et al., 1997) although their significance in viral replication is unclear.
The first one is located near the N terminus of NS4B protein and its cleavage
was observed only when the NS4A/NS4B cleavage was blocked (Kolykhalov et
al., 1994). The second one, in the middle of NS5A protein, was seen in cell-free
proteolysis experiments (Markland et al., 1997).

HCV NS4A PROTEIN IS A COFACTOR ESSENTIAL FOR ACTIVITY OF THE

NS3 SERINE PROTEASE
It was demonstrated during these trans-cleavage and processing kinetics studies
that an additional HCV-encoded protein, NS4A, is required as an activating
cofactor for the optimal activity of the NS3 serine protease (Bartenschlager et al.,
1994; Bartenschlager et al., 1995b; Failla et al., 1994; Lin et al., 1994; Tanji et al.,
1995). In the absence of NS4A protein, only the NS5A/NS5B site, but not the other
three cleavage sites (NS3/NS4A, NS4A/NS4B, and NS4B/NS5A), was partially
processed by the NS3 serine protease alone. The presence of NS4A not only enables
efficient processing at these three junctions and but also resulted in enhancement
of the NS5A/NS5B cleavage. Deletion analysis showed that the central region
(residues 21 to 34) of the NS4A (Fig. 2B), a 54-residue protein, is essential and
sufficient for the cofactor function of the NS3 serine protease (Bartenschlager et
al., 1995b; Failla et al., 1995; Lin et al., 1995; Satoh et al., 1995; Tanji et al., 1995).
In addition, NS4A forms a non-covalent complex with the NS3 serine protease,
which was stable in the presence of non-ionic detergent (Bartenschlager et al.,
1995b; Failla et al., 1995; Hijikata et al., 1993a; Hijikata et al., 1993b; Lin et al.,
1995; Satoh et al., 1995). Deletion studies indicate that the N-terminal 22 residues
of NS3 and the above-mentioned central region of NS4A is involved in interaction
between two proteins (Bartenschlager et al., 1995b; Failla et al., 1995; Koch et
al., 1996; Lin et al., 1995; Satoh et al., 1995; Tanji et al., 1995). Substitutions that
disrupted the interaction between NS3 and NS4A also resulted in reduction or loss
of protease activity, suggesting that formation of an NS3-NS4A complex could be
a pre-requisite for a functional serine protease (Butkiewicz et al., 1996; Koch et
al., 1996; Lin et al., 1995; Shimizu et al., 1996; Steinkühler et al., 1996a; Tomei
et al., 1996).

NS3 SERINE PROTEASES AND THEIR COFACTORS IN THE FLAVIVIRIDAE

FAMILY
The activation of a virus-encoded protease by peptide(s) derived from another viral
protein is not uncommon for viruses. This phenomenon seems to be a common
feature of members of Flaviviridae family. As mentioned earlier, comparative
sequence analysis suggests the presence of a chymotrypsin-like serine protease

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in the N-terminal one-third of NS3 protein of flaviviruses, pestiviruses, and HCV,

which accounts for all three known genera of the Flaviviridae family at that time
(Bazan and Fletterick, 1989; Bazan and Fletterick, 1990; Gorbalenya et al., 1989a;
Miller and Purcell, 1990). A chymotrypsin-like serine protease was later identified
in the newly discovered 4th genus of this family, hepatitis G virus (HGV) or GB
virus (Leary et al., 1996; Scarselli et al., 1997). In each member of the Flaviviridae
family that has been studied so far, a virus-encoded cofactor is needed to activate
the corresponding NS3 serine protease. It is the NS4A protein, located immediately
downstream of the NS3 protein, in the case of HCV, bovine viral diarrhea virus
(BVDV) (Wiskerchen and Collett, 1991; Xu et al., 1997) of pestiviruses, and HGV
or GB virus (Butkiewicz et al., 2000; Sbardellati et al., 2000). For flaviviruses, it
is the NS2B protein, located immediately upstream of the NS3 protein, that fulfills
the role of activator for the corresponding NS3 serine protease of yellow fever virus
(Chambers et al., 1991) or dengue virus (Cahour et al., 1992; Falgout et al., 1993;
Falgout et al., 1991). Again, a short peptide corresponding the central region of
the NS4A cofactor of BVDV (Tautz et al., 2000) or GB virus (Butkiewicz et al.,
2000), or a central 40-residue fragment of the dengue NS2B protein (Falgout et
al., 1993) was sufficient for activation of the corresponding NS3 serine protease.
It should be noted that there is little sequence homology between different genera
with regard to these activators of the NS3 serine proteases. In fact, the NS3 serine
proteases of different genera of the Flaviviridae family have quite distinct specificity
for substrates, especially on the P1 residues: Cys or Thr for HCV (Grakoui et al.,
1993a) and HGV or GB virus (Scarselli et al., 1997), Leu for pestiviruses (Xu et
al., 1997), and Lys or Arg for flaviviruses (Chambers et al., 1991).

ADENOVIRUS PROTEASE HAS THREE ACTIVATING COFACTORS

The examples of virus-encoded proteases and their activating cofactors are not
limited to Flaviviridae family. In fact, the very first example was discovered in
human adenovirus. A cysteine protease encoded by human adenovirus (AVP) (for a
review see Mangel et al., 2003) was found in virions and activated by an 11-residue
peptide cofactor from the C-terminus of pVI protein (pVIc) (Mangel et al., 1993;
Webster et al., 1993). In contrast to the HCV serine protease, the pVIc cofactor is
covalently linked to the AVP by a disulfide bond (Ding et al., 1996). This protease
requires binding of not one, but two co-factors, pVIc and adenovirus DNA for the
optimal activity (Baniecki et al., 2001; Mangel et al., 1993; Webster et al., 1993).
One possible explanation for the DNA cofactor is that AVP moves along the viral
DNA looking for precursor protein cleavage sites much like RNA polymerase
moves along DNA looking for promoter (McGrath et al., 2001). In addition, actin,
a cellular cytoskeletal protein, was found to activate AVP as well (see below).

VIRUS-HOST INTERACTION OF HCV NS3-4A SERINE PROTEASE

Several recent studies suggest that HCV NS3-4A protease could be one of the
weapons that HCV uses to breakdown the host antiviral response (reviewed in

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chapter 13). Innate immune response is trigged upon recognition by a family of

toll-like receptor (TLR) of certain pathogen-associated molecular patterns. TLR-3
binds one of these patterns, dsRNA, a replication intermediate of many viruses,
and initiates a massive type-I IFN-mediated antiviral response through activation
of IFN regulatory factor 3 (IRF-3), a transcription activator. Recently, it was shown
that binding of dsRNA to two DExD/H box RNA helicases, retinoic acid inducible
gene I (RIG-I) and Helicard (Mda5) in cytoplasm induces a TLR-3-independent
IFN response pathway, through activation of IRF-3 and NF-κB. The fact that the
Huh7 hepatoma cell line, which is deficient in TLR-3-dependent pathway, became
highly permissive for replication of HCV replicon RNA when an inactivating
mutation in the RIG-I gene was selected in a subclone, Huh7.5 (Sumpter et al.,
2005) suggests that both pathways play a role in anti-HCV immune responses. It
was reported that expression of active HCV NS3-4A protease blocked activation
of both TLR-3-dependent (Foy et al., 2003) and TLR-3-independent (Foy et al.,
2005) signal transduction cascades, and therefore prevented the IFN-induced
antiviral response against HCV RNA replication. In the case of TLR-3-dependent
pathway, the substrate of HCV NS3-4A protease was reported to be Toll-IL-1
receptor domain-containing adaptor inducing IFN-β (TRIF or TICAM-1) (Li et
al., 2005). TRIF, a critical player in the TLR-3-dependent pathway, recruits two
kinases, TBK1 and IKKε, to phosphorylate and activate IRF-3. In the case of
TLR-3-independent cascade, the HCV NS3-4A protease was reported to cleave
Cardif, a new CARD-domain containing adaptor protein that interacts with RIG-I
and recruits IKKα, IKKβ, and IKKε kinases, resulting in activation of IRF-3 and
NF-κB (Meylan et al., 2005).

Interference of host function by virus-encoded proteases is not limited to HCV.

Actin, a cellular cytoskeletal protein, was found to interact and activate AVP (Brown
et al., 2002). More specifically, it is an 11-residue peptide corresponding to the C
terminus of actin, which is highly homologous to pVIc, was shown to bind and
stimulate AVP activity (Brown and Mangel, 2004). The activation of AVP by actin
has been proposed to be a mechanism for adenovirus to facilitate the cleavage of
cytoskeletal proteins, preparing the infected cells for lysis and release of nascent
virions (Brown and Mangel, 2004). In the presence of actin, the cellular skeletal
protein, AVP is activated and then is able to mediate proteolysis of cytokeratin
18, another cytoskeletal protein and, not surprisingly, actin itself (Brown et al.,
2002).

THREE DIMENSIONAL STRUCTURES

A DOUBLE β-BARREL FOLD OF THE HCV NS3-4A SERINE PROTEASE

In 1996, two groups presented seminal studies showing the X-ray structures of the
HCV NS3 serine protease domain (Kim et al., 1996; Love et al., 1996). Kim et al.

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solved an X-ray structure of NS3 serine protease domain (residues 1 to 181) of the
HCV H strain of genotype 1a in a non-covalent complex with a NS4A cofactor
peptide (residues 21 to 39) (Kim et al., 1996). The HCV serine protease forms a
double-barrel fold (Fig. 3A), which is similar to that of serine proteases from the
chymotrypsin/trypsin super-family. The catalytic triad is located in a cleft between

Fig. 3. X-ray structures of the HCV NS3-4A serine protease.

(A) Ribbon diagram of the NS3 protease domain in a complex with an NS4A cofactor peptide. The
N-terminal sub-domain of the NS3 protease (in green), including the NS4A β-strand (in magenta),
is on the left side and the C-terminal sub-domain (in green) on the right side. Shown in ball-and-
stick are the catalytic triad, His57, Asp81, and Ser139, at the top, and the zinc atom (in cyan), which is
tetrahedrally coordinated by three Cys residues (Cys97, Cys99, and Cys145) (in yellow spheres) and,
via a water molecule (in red), His149 (not shown), at the bottom.

(B) A close-up view of the zinc-binding site. Shown in ball-and-stick presentation is the zinc atom
(in cyan), which is coordinated by three Cys residues (Cys97, Cys99, and Cys145) (in yellow spheres)
and, via a water molecule (W, in red sphere), His149.

(C) Stick diagram of interaction between the two N-terminal β-strands of the NS3 serine protease
domain (thin bonds) and the NS4A activating cofactor (thick bonds). Several residues in the central
region of NS4A cofactor, including Val23, Ile25, Ile29, and Leu31, make extensive hydrophobic
interaction with many hydrophobic side chains of these two β-strands of the NS3 protease that form
a "sandwich" with the NS4A β-strand. NS4A also forms numerous main-chain hydrogen bonds with
these NS3 residues. (Reprinted from Cell (1996) 87: 343-355, J.L. Kim et al., Crystal structure of
the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. With
permission from Elsevier.)

A colour version of this figure is printed in the colour plate section at the back of this book.

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 HCV NS3-4A Serine Protease

B C

two sub-domains (or barrels), with His57 and Asp81 in the N-terminal sub-domain
and Ser139 in the C-terminal one. The C-terminal sub-domain (residues 96–180)
contains the conventional six-stranded β barrel, common to most members of the
chymotrypsin family, followed by a structurally conserved α helix. The N-terminal
sub-domain (residues 1-93) consists of eight β-strands, including seven from the
NS3 protein (Fig. 3A, colored in green) and one from the NS4A peptide (Fig. 3A,
colored in magenta), and the latter one is sandwiched between two β-strands of the
N-terminal sub-domain of NS3. In addition, the C-terminal sub-domain contains
a tetrahedrally coordinated metal ion, presumably a zinc atom, located at one end
of the β barrel, opposite to the catalytic triad.

EFFECTS OF NS4A BINDING

The extensive interaction between NS3 and NS4A results in a tightly packed β-barrel
and buries an additional 2400 Å2 of surface area of the NS3 protease (Fig. 3C). All
but two of the main-chain carbonyl and amide groups of the NS4A residues 23–31
(Fig. 2B) form hydrogen bonds with NS3. Hydrophobic side chains of several
conserved NS4A residues (Val23, Ile25, Ile29, and Leu31) (Fig. 2B) are buried in
hydrophobic core of the N-terminal sub-domain of NS3 (Kim et al., 1996) (Fig.
3C). These observations in the X-ray structure confirm the previous deletion and
mutagenesis studies, in which the central region of NS4A (residues 21–33), and

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in particular, the several conserved hydrophobic residues (Ile25, Ile/Val29), were

shown to be critical for optimal binding of NS4A to the NS3 serine protease and
activation of the protease activity (Butkiewicz et al., 1996; Lin et al., 1995; Shimizu
et al., 1996; Tanji et al., 1995). The mechanism of activation by the NS4A cofactor
on the NS3 serine protease activity were best illustrated by a direct comparison
of three X-ray structures of HCV NS3 protease domain. In an X-ray structure of
uncomplexed NS3 serine protease domain (residues 1 to 189) of the HCV BK
strain of genotype 1b, solved in the absence of an NS4A cofactor, the N-terminal
sub-domain of NS3 protease has only six β-strands and the N-terminal 28 residues
of NS3 are extending away from the core of the protein as a flexible loop (Love
et al., 1996). In two X-ray structures of the NS3 protease-NS4A cofactor complex
of HCV H strain (Kim et al., 1996) or BK strain (Yan et al., 1998), respectively,
the first 28 NS3 residues fold into an α-helix and a β-strand. This additional NS3
β-strand is spatially located next to the β-strand of NS4A, and both contribute to
the formation of an eight-strand β-barrel (Fig. 3A). These observations in X-ray
structures are consistent with earlier results that deletion of the N-terminal 22
residues of NS3 resulted to loss of the NS4A binding (Bartenschlager et al., 1995b;
Failla et al., 1995; Koch et al., 1996; Satoh et al., 1995). The more striking effect of
NS4A binding is on the orientation of the catalytic triad, which is highly conserved
in the chymotrypsin serine protease family so that the Asp residue stabilizes the
His residue after the imidazole ring of His deprotonate the OH group of the Ser
nucleophile. In the absence of the NS4A cofactor, the conformation of the triad is
significantly distorted so that the protease is not expected to have an appropriate
activity, as observed in biochemistry studies. In the X-ray of NS3 serine protease
domain alone, the imidazole ring of His57 is located too far away to effectively
deprotonate the nucleophilic OH of Ser139 or to be stabilized by the Asp81 (Love
et al., 1996). In the presence of NS4A cofactor peptide, the catalytic triad is in the
characteristic position expected for a chymotrypsin-like serine protease (Kim et
al., 1996; Yan et al., 1998).

Although both HCV serine protease and adenovirus cysteine protease require the
binding of the corresponding activator(s) to be fully functional, the mechanisms of
cofactor binding and activation are quite different. In the X-ray structure of human
adenovirus-2 protease in a complex with its pVIc cofactor, the catalytic triad of
AVP (Cys-His-Glu) adapted an arrangement similar to that of papain, and the pVIc
cofactor extends a β-sheet, which is distant from the active site (Ding et al., 1996).
Another difference is that pVIc binds to AVP by covalent disulfide bond and forms
a 6th strand on the β-sheet (McGrath et al., 2003).

ZINC-BINDING SITE
Alignment of amino acid sequences showed that three Cys and one His residues
are well conserved in the NS3 protease domain of various HCV strains (Fig. 2A)

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and closely related GB viruses or hepatitis G viruses. Computational modeling

analysis suggests that these four residues could form a zinc-binding site, which
is located opposite to the catalytic triad in the NS3 serine protease model (Failla
et al., 1996). This prediction was quickly confirmed by both X-ray structural and
biochemical studies. In the X-ray structure of the NS3 serine protease domain, a
zinc ion is tetrahedrally coordinated by four residues, Cys97, Cys99, Cys145, and
through a water molecule, His149 (Fig. 3B) (Kim et al., 1996; Love et al., 1996).
Substitution of any of the three Cys residues involved in zinc-binding with Ala led
to significantly reduced protease activity, whereas mutation at His149 had much less
effect (Hijikata et al., 1993a). This zinc-binding pocket is located in a cleft between
two β-barrels and at the opposite end of the catalytic triad (Fig. 3A). The zinc atom
is at least 20 Å away from the catalytic Ser139, suggesting the coordination of a zinc
atom plays more a structural role rather than a catalytic function, as in case of several
other viral proteases, including poliovirus and rhinovirus 2A cysteine proteases
(Sommergruber et al., 1994; Voss et al., 1995; Yu and Loyd, 1992). A similar
arrangement of Cys and His residues, as in Cys-X-Cys……Cys-X-His, where X is
any residue, is also found in the cysteine protease 2A of the Picornavirae family.
In these picornavirus 2A cysteine proteases, a tightly bound zinc atom is found to
be critical for integrity and stability of the properly folded protein structure, rather
than proteolysis. In many other serine proteases, such as chymotrypsin, a disulfide
bond is found in a similar location, suggesting the zinc-binding pocket in the HCV
NS3 serine protease plays a similar role as the disulfide bond in stabilizing the
relative position of two β-barrels. Additional confirmation came from biochemical
experiments, which showed that the purified, active HCV NS3 protease domain
contained an equimolar amount of zinc (De Francesco et al., 1996; Stempniak et
al., 1997). NS3 protein expressed in the absence of zinc in E. coli was not folded
properly and therefore deficient of the protease activity (De Francesco et al., 1996;
Stempniak et al., 1997).

THE SUBSTRATE BINDING GROOVE

The HCV NS3-4A serine protease retains some highly conserved features of the
chymotrypsin family, such as spatial location of the catalytic triad of His57, Asp81,
and Ser139, as well as the positions of backbone amides of Gly137 and Ser139,
which forms the oxyanion hole (Kim et al., 1996). A twisted strand (residues
Arg155-Ala156-Ala157-Val158) of the HCV protease superimposes well with the
corresponding strand of residues in other serine proteases in the chymotrypsin
family, which makes hydrogen bonds with the P3 carbonyl and the P1 and P3
amides of peptidomimetic inhibitors of serine proteases (Edwards and Bernstein,
1994). However, the HCV serine protease does display some significant difference
from other serine proteases, such as chymotrypsin. For examples, several loops that
interact with P4, P3, and P2 moieties of inhibitors and form a well-defined substrate-
binding pocket in many other serine proteases are either shortened significantly or

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deleted in the HCV serine protease. Lack of side chain interaction is compensated
by an extensive network of hydrogen bonds between the main chain atoms of
the protease and substrate. However, the absence of these long loops results in a
shallow, solvent-exposed substrate-binding groove and renders the design of small-
molecule, peptidomimetic inhibitors against the HCV NS3-4A serine protease an
extremely challenging task.

THE S1 POCKET
Sequence alignment of four cleavage sites in the HCV polyprotein indicates that
there are three conserved positions in the HCV NS3-4A protease substrates: an Asp
or Glu at P6, a Cys or Thr at P1, a Ser or Ala at P1' (Fig. 2C). Three of the cleavage
sites, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B, have a Cys at the P1 position,
while the NS3/NS4A site has a P1 Thr residue. In the X-ray structures, the S1 pocket
is primarily determined by three hydrophobic residues, Leu135, Phe154, and Ala157
(Kim et al., 1996; Love et al., 1996). Phe154 is located at the bottom of the S1 pocket
and clearly in position to make favorable van der Waals interactions with the P1
side chain. The small and hydrophobic nature of this S1 pocket is complementary
to a relatively small and lipophilic side chain of a Cys. In addition, it is known that
the sulfhydryl group of a Cys residue forms a favorable electrostatic interaction
with the aromatic ring of Phe (Burley and Petsko, 1988). Proteolysis of substrates
with larger P1 residues was allowed when Phe154 was substituted with a residue
that has a smaller side chain, such as Thr, along with replacement of Ala157 with a
Gly, which altered the specificity of the mutated NS3 protease (Failla et al., 1996;
Koch and Bartenschlager, 1997).

THE S6 POCKET
All HCV NS3-4A cleavage sites contain an acid residue (usually Asp) at the P6
position (Fig. 2C). The P6 acid residue, sometimes along with an acidic residue
at the P5 position, is believed to form electrostatic interactions with a cluster of
positively charged residues of the NS3 protease, Arg123, Arg161 and Lys165 (Koch
et al., 2001; Steinkühler et al., 2001).

ASSAYS FOR THE HCV NS3-4A SERINE PROTEASE

EVOLUTION OF IN VITRO BIOCHEMICAL ASSAYS

While cell-based expression and proteolytic processing experiments were useful
for providing the first glimpses of the activity of the HCV NS3-4A serine protease,
their limitations were quickly reached given the complexity of cellular environment.
Further understanding of the functions of this protease and substrate, which would
enable meaningful drug discovery efforts, required establishment of efficient
biochemical assays. In the early-generation cell-free trans-cleavage assays, the
protease, the substrate or both were derived from cell lysates or in vitro translation,
which may or may not have been coupled with in vitro transcription (Bouffard et al.,

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1995; Hahm et al., 1995; Koch et al., 1996; Lin and Rice, 1995; Suzuki et al., 1995;
Tomei et al., 1996). Later, in vitro translated polyprotein substrates were used for
monitoring activity of purified NS3 protease, which was generated as recombinant
proteins in E. coli, baculovirus, or yeast expression systems (Butkiewicz et al., 1996;
D'Souza et al., 1995; Markland et al., 1997; Steinkühler et al., 1996a). However,
these assays using in vitro translated polyproteins are much less quantitative than
those using synthetic peptides as substrates, which are cleaved by the HCV serine
protease and the products are analyzed on high performance liquid chromatography
(HPLC) (Bianchi et al., 1996; Inoue et al., 1998; Kakiuchi et al., 1995; Landro et
al., 1997; Shimizu et al., 1996; Steinkühler et al., 1996a; Steinkühler et al., 1996b;
Sudo et al., 1996; Urbani et al., 1997; Zhang et al., 1997). A colorimetric substrate,
EDVVαAbuC-p-nitroanilide (5A-pNA), in which the P2 Cys was substituted with
Abu and the whole P'-side replaced with a colorimetric leaving group, pNA, was
used to increase assay convenience (Landro et al., 1997). It was observed when the
scissile amide bond was replaced with an ester linkage, which allows ready trans-
esterification of the scissile bond to the acyl-enzyme intermediate, these substrates
displayed much improved catalytic efficiency (kcat/Km), allowing detection of
activity with sub nM of NS3 protease (Bianchi et al., 1996). An internally quenched
fluorogenic donor/acceptor couple, based on resonance energy transfer, was then
incorporated into a depsipepetide substrate, which was shown to be suitable for
high-throughput screening (Taliani et al., 1996).

ROLE OF NS4A COFACTOR

It was shown in these studies that a synthetic peptide corresponding to the central
region (residues 21–34) of NS4A could be used to substitute for the full-length
NS4A protein in activation of the HCV NS3 serine protease domain (Butkiewicz
et al., 1996; Lin et al., 1995; Shimizu et al., 1996; Steinkühler et al., 1996a; Tomei
et al., 1996). Substitution study of NS4A peptide indicates that the following
residues of NS4A, Val23, Gly27, Arg28, and in particular, Ile25 or Ile29 are critical
for its cofactor function (Butkiewicz et al., 1996; Koch et al., 1996; Lin et al.,
1995; Shimizu et al., 1996; Tomei et al., 1996). The activator function of NS4A
peptide was largely due to an increase in the turnover rate (kcat) with a small or
little change in substrate affinity (Km) for substrates corresponding to all three
cleavage sites that can be cleaved in trans, namely, NS4A/NS4B, NS4B/NS5A,
and NS5A/NS5B (Landro et al., 1997; Shimizu et al., 1996; Steinkühler et al.,
1996a; Steinkühler et al., 1996b). The increase is more dramatic (>100-fold) for
the less efficient substrate, the NS4B/NS5A substrate (Steinkühler et al., 1996b).
An 1:1 stoichiometric amount of NS3 protease domain protein and NS4A peptide
is sufficient for the maximal activation, suggesting the active form of HCV serine
protease is a heterodimer (Steinkühler et al., 1996b). The dissociation constant (kd)
was determined to be in low µM range, but the association rate (kon) was very low,
suggesting conformational transitions to be the rate limiting event for the formation
of NS3-4A protease complex (Bianchi et al., 1997).

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SUBSTRATE SPECIFICITY
In enzymatic assays, a decapeptide substrate, with 6 residues on the P-side and 4
more on the P'-side, was found to be optimal for efficient proteolysis by the HCV
NS3-4A protease. Further truncation from either the P-side or the P'-side resulted
in a significantly drop in catalytic efficiency (Landro et al., 1997; Steinkühler et al.,
1996a; Zhang et al., 1997). Sequence alignment of natural decapeptide substrates
for the HCV NS3-4A serine protease revealed a conserved acidic residue (Asp
or Glu) at the P6, a Cys or Thr at the P1, and a Ser or Ala at the P1', resulting
in the consensus substrate sequence of (Asp/Glu)–X–X–X–X–(Cys/Thr)↓(Ser/
Ala)–X–X–X, whereas X indicates an variable residue (Grakoui et al., 1993a).
In cell culture transfection experiments, it was found that the P6 acidic residue
was dispensable and substitutions at the P1' were reasonably tolerated. However,
most mutations introduced at the P1 inevitably resulted in a significant loss of
proteolytic processing, indicating that the P1 Cys or, to a less extent, Thr, is the
major determining factor for substrate recognition (Bartenschlager et al., 1995a;
Kolykhalov et al., 1994; Komoda et al., 1994; Leinbach et al., 1994; Tanji et al.,
1994a). The preference for the peptide substrate sequence in enzyme assay is also
reminiscent of the consensus sequences of the HCV natural polyprotein substrates.
The optimal peptide substrate in enzyme assay has an acidic residue (Glu or Asp)
at P6, a Cys at P1, and a Ser or Ala at P1' (Landro et al., 1997; Urbani et al., 1997;
Zhang et al., 1997). The other residues besides these three key positions (P6, P1, and
P1') may also play roles in recognition by the NS3-4A serine protease, as evidenced
by the drastically different catalytic efficiency in the following order: NS5A/NS5B
> NS4A/NS4B >> NS4B/NS5A (Landro et al., 1997; Steinkühler et al., 1996b).
These differences in catalytic efficiency are consistent with the processing order
and polyprotein intermediates observed in cell culture.

CROSS TALK BETWEEN THE PROTEASE AND HELICASE DOMAINS OF

NS3?
In Flaviviridae family, the NS3 protein is invariably an at least bi-functional
protein, with the serine protease in the N terminal one-third and the helicase in the
C-terminal two-thirds. It is not unreasonable to suggest that there may be cross
communication between these two enzyme functions residing in the same protein.
Indeed, polynucleotides, especially poly(U), which are stimulants for the ATPase
activity of the NS3 helicase, also enhanced the serine protease activity in a full-length
NS3-4A protein complex purified from over-expressing COS cells (Morgenstern et
al., 1997). Because the poly(U) did not stimulate the protease activity of the purified
NS3 serine protease domain, these results suggest that poly(U) enhances the protease
activity of the full-length NS3-4A complex through its interaction with the helicase
domain, and the latter then interacts and stimulates the protease domain. The presence
of the serine protease domain seems to be required for optimal binding of poly (U)
to the helicase domain because the dissociation constant, Kd, of poly(U) was 10-fold
lower against full-length NS3 protein than that against the helicase domain alone

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(Kanai et al., 1995). However, another study showed that poly (U) inhibited the
protease activity of both protease domain alone and full-length NS3, in the presence
of a synthetic NS4A cofactor peptide (Gallinari et al., 1998).

BIOCHEMISTRY OF ADENOVIRUS PROTEASE, AVP

The pVIc peptide also forms a 1:1 complex with the adenovirus protease, AVP, and
enhances its catalytic efficiency by hundreds fold, while the addition of adenovirus
DNA increased the kcat/Km even further. Both cofactors enhance AVP activity by
increasing kcat, not by decreasing Km (Baniecki et al., 2001; Mangel et al., 1996;
McGrath et al., 2001). These two cofactors bind to the AVP with a dissociation
constant (Kd) in the µM range, 4.4 µM for pVIc and 0.09 µM for a 12-mer ssDNA
(Baniecki et al., 2001). On the other hand, actin binds to the adenovirus protease
AVP with a much lower equilibrium dissociation constant, 4.2 nM, than those
exhibited by two viral, nuclear cofactors for AVP, the 11-amino acid peptide pVIc
and the viral DNA (Brown and Mangel, 2004). The catalytic efficiency, kcat/Km,
for substrate hydrolysis by AVP increased 150,000-fold in the presence of actin
(Brown and Mangel, 2004).

A DRUG DISCOVERY TARGET

Ever since its identification in 1993, the HCV NS3-4A serine protease has been
subjected to intense efforts on the discovery of potent, selective inhibitors as
potential new therapies for the hepatitis C patients. As described above, numerous
milestones on essential tools for drug discovery efforts, including biochemical
and cellular assays, determination of X-ray structures, and animal models, have
been achieved by many laboratories around the world during this campaign. The
success of HIV protease inhibitor drugs demonstrates that viral proteases, such as
the HCV NS3-4A serine protease, could be excellent targets for a structure-based
drug design approach. Indeed, rational drug design has been used successfully
for discovery of potent, selective inhibitors of other viral proteases, such as
cytomegalovirus (CMV) proteases or rhinovirus proteases. However, in the case
of HCV NS3-4A serine protease, design efforts for a small-molecule, orally
available, potent, and selective drug candidate were partially hampered by the
shallow, remarkably hydrophobic, substrate-binding groove of the HCV protease.
Nevertheless, significant progress has been made in recent years to identify potent
small-molecule inhibitors against the HCV protease. The ensuing discussion will
focus on discovery of active site, peptidomimetic inhibitors, while other types of
inhibitors will be briefly described.

NON-COVALENT, PRODUCT-BASED INHIBITORS

In 1998, two groups presented critical findings that, after an oligopeptide
corresponding to the NS4A/NS4B or NS5A/NS5B substrate was cleaved by the
HCV NS3 protease in the presence of the NS4A cofactor, the C-terminal cleavage

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product quickly dissociated from the enzyme but the N-terminal product was
released rather slowly. This resulted in feedback inhibition by the N-terminal
cleavage product, i.e., the hexa-peptide from the C terminus of the NS4A or NS5A,
respectively (Llinas-Brunet et al., 1998b; Steinkühler et al., 1998). In addition, the
free carboxylic group of the P1 residue, which is liberated by the cleavage of the
substrate peptide bond, was recognized as an essential feature imparting selectivity
with respect to other serine proteases (Llinas-Brunet et al., 1998a). The X-ray
structure of a full-length NS3-4A protein provided an excellent explanation for the
observed product-based inhibition (Yao et al., 1999). In the X-ray structure, the
last residue of the NS3 protease, Thr631, which is the P1 residue of the NS3/NS4A
cis-cleavage site, was bound in the active site of the NS3 serine protease domain.
Apparently, after the NS3 serine protease cleaves the NS3/NS4A peptide bond,
the N-terminal cleavage product, with the Thr631 as the C-terminal residue, is not
released from the NS3 protease in the X-ray structure. With this seminal finding,
two groups presented extensive structure-and-activity relationship (SAR) results
using the hexa-peptide from the C terminus of the NS4A or NS5A, respectively.
These SAR studies demonstrated that a combination of a thiol-containing residue
at the P1 position and acidic residues at the P5–P6 positions is required for optimal
binding and resulted in potent hexa-peptide inhibitors (Ingallinella et al., 1998;
Llinas-Brunet et al., 2000). The preference displayed by NS3 protease for a thiol-
containing residue, such as Cys, as the P1 anchor results from the shape of the S1
pocket. This small, hydrophilic pocket, lined by the hydrophobic residues of Val132,
Leu135, and Phe154, is complementary to the small and hydrophilic side chain of
Cys (Kim et al., 1996). In addition, the sulfhydryl (SH) group of the P1 Cys can
interact in a unique way with the aromatic ring of Phe154. The second anchor of the
hexa-peptide inhibitors is the pair of P5–P6 acidic residues, which is thought to form
electrostatic interactions with a cluster of basic amino acids of the NS3 protease,
including Arg123, Arg161, and Lys165 (Di Marco et al., 2000; Koch et al., 2001).

The presence of a thiol-containing side chain in the P1 position presents a major

hurdle for the chemical synthesis and stability of potential inhibitors, and therefore,
limits its use in clinical development (Emery et al., 1992). Substitution of P1
Cys with amino acids containing small hydrophobic side chains, such as Ala or
α-aminobutyric acid, resulted in a decline in inhibitory potency due to the sub-
optimal filling of the P1 subsite. Replacement with amino acids containing larger
hydrophobic side chains led to a significant loss of activity presumably due to steric
hindrance (reviewed in Steinkühler et al., 2001). It was shown that a difluoromethyl
group is an effective mimetic of the thiol, likely due to the similarity of their steric
and electrostatic properties (Narjes et al., 2002a). In addition, 1-aminocyclopropy
lcarboxylic acid was shown to be an effective surrogate as well, with little impact
on potency when it replaced the natural P1 Cys in a hexa-peptide inhibitors (Llinas-
Brunet et al., 2000).

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While the P5–P6 acidic residue pair contributes significantly to the binding of hexa-
peptide inhibitors to the NS3-4A protease, it does bring two major disadvantages
that render the hexa-peptide inhibitors not suitable for clinical development as oral
drugs: negative charges and a rather large molecular weight (over 1,000 Daltons).
The negative charges of the P5–P6 acidic pair would most likely prevent the hexa-
peptides from penetrating into cells, which is reflected in the lack of cellular potency
of these inhibitors in HCV replicon cells. Removal of the P5 and P6 acidic residues
resulted in, as expected, a significant loss in potency of tetra-peptide inhibitors,
which has to be compensated by improvement in other subsites of the inhibitors.
Significant enhancement in potency was achieved with the addition of large,
hydrophobic aromatic rings to the P2 Pro group, resulting in potent tetra-peptide
inhibitors (Goudreau et al., 2004b). In addition, a macrocyclic ring was designed
to link the side chain of the P1 and P3 residues to reduce the peptidic nature and
provide rigidity to pre-order the binding conformation (Tsantrizos et al., 2003).
The rigidity imparted by the ring structure constricts the molecule into exclusively
adopting the correct rotamer for binding to the backbone of the NS3 protease. A 15-
membered ring macrocycle was found to be optimal (Goudreau et al., 2004a). All
these efforts, coupled with the previously described aminocyclopropane carboxylic
acid at P1 (Rancourt et al., 2004), resulted in identification of a clinical candidate,
BILN 2061 (ciluprevir) (Fig. 4A) (Llinas-Brunet et al., 2004; Tsantrizos, 2004).
This compound has an excellent potency against the HCV NS3-4A serine protease,
with estimated Ki values of 0.66 nM and 0.30 nM against the genotype 1a and 1b
HCV protease, respectively (Lamarre et al., 2003). Treatment of the genotype 1a
and 1b HCV replicon cells with BILN 2061 for 3 days resulted in a dose-dependent
decrease of HCV RNA with a mean IC50 of 4 nM and 3 nM, respectively (Lamarre
et al., 2003).

REVERSIBLE, COVALENT INHIBITORS

The prospect of the hexapeptide inhibitors of the HCV NS3-4A protease being
developed into potential oral therapies is rather low because of their large molecular
weight and the presence of multiple carboxylic acids, both of which are major
obstacles for achieving high oral bioavailability. Another approach to compensate
for the loss of P5–P6 acidic pair is to design a "warhead" that forms covalent bonds
to the catalytic Ser nucleophile, or so-called serine-trap. Ideally, these serine-trap
inhibitors will form a covalent bond to the catalytic Ser139, but this covalent bond
will be cleaved so that the inhibition is not irreversible against the proteases. In the
early stages of research on covalent HCV protease inhibitors, most of the standard
serine-trap warheads, such as α-haloketones or heterocyclic ketones, displayed
poor inhibition of this enzyme (Perni et al., 2004b). Although aldehydes were
useful tools for SAR (Perni et al., 2003a), the inherent instability of aldehydes, in
particular, aliphatic aldehydes, which are oxidized, rendered this warhead unsuitable
for further development. The sulfonamido group is also an attractive and effective

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B C

Fig. 4. Chemical diagrams of the HCV NS3-4A protease inhibitors.

(A) Chemical structures of the natural HCV NS5A/NS5B deca-peptide substrates (from P6 to P4'),
and two NS3-4A protease inhibitors, BILN 2061 and VX-950.

(B) Schematic and (C) X-ray structure of the α-ketoamide motif bound to the NS3-4A protease
active site. The inhibitor is shown at the left and the protease at the right. Two hydrogen bonds in the
oxyanion hole were indicated with dashed lines. In the X-ray structure, nitrogens are shown in blue,
oxygens in red, and hydrogen in white. The resolution of this X-ray structure is 2.9 Å.

A colour version of this figure is printed in the colour plate section at the back of this book.

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warhead for peptidomimetic scaffolds (Johansson et al., 2003). One of boronate

esters demonstrated very strong, but presumably reversible, binding to the enzyme
(Ki = 8 nM) (Priestley et al., 2002).

Another potent warhead is the α-ketoacid, which is capable of both covalent

attachment to the catalytic Ser139 and electrostatic attraction of the carbonyl
terminus (Colarusso et al., 2002). The most potent inhibitors in a dipeptide series
with the α-ketoacid warhead inhibited the HCV serine protease with an IC50 of
3 µM in enzyme assay (Nizi et al., 2002). Diketones and α-ketoamides are also
covalent but reversible functionalities that could serve as stable, reversible and
effective binding groups (Han et al., 2003; Perni et al., 2004b), although in general,
diketones are less potent than the corresponding α-ketoamides (Han et al., 2000).
The covalent attachment of α-ketoamide group with Ser139, as well as an unusual
interaction between the dicarbonyl motif and the oxyanion of the protease (Fig.
4B), provide exceptional potency against the HCV NS3-4A protease. This class
of inhibitors has a slow-binding mechanism of action due to the requirement of an
unusual re-arrangement in the active site of the NS3 similar to that observed for
ketoacids (Liu et al., 2004; Perni et al., 2004b). Extensive optimization of the P3
and P4 hydrophobic groups, removal of the acidic charge at P1' and, finally, the
modification of the P2 Pro substituent into a bicyclic Pro motif (Perni et al., 2003a;
Perni et al., 2004a; Perni et al., 2004b; Sun et al., 2004; Victor et al., 2004; Yip et
al., 2004a; Yip et al., 2004b) resulted in identification of a clinical candidate, VX-
950 (Fig. 4A) (Perni et al., 2003b). While the optimal length for recognition by the
HCV NS3-4A protease is 10 amino acids in natural HCV substrates, the backbone
of these inhibitors was truncated to a tetrapeptide scaffold while maintaining
significant binding affinity for the NS3-4A serine protease (Perni et al., 2003b).
This compound had excellent potency against the HCV NS3-4A serine protease,
with a Ki* value of 7 nM against the genotype 1a HCV protease (Perni et al.,
2003b). Despite of the large difference in the IC50 values in a 2-day replicon cell
assay, these two inhibitors had a comparable ability to induce a 4-log10 reduction
of HCV RNA levels after an extended incubation with replicon cells (Lin et al.,
2003; Lin et al., 2006).

NON-COVALENT SUBSTRATE MIMETICS AND P' INHIBITORS

To date, most peptidomimetic, active site inhibitors of the HCV serine protease
were designed against those binding pockets on the P-side, and much less effort
has been spent on the P'-side. It was found that the replacement of the P1' residue
of a decapeptide substrate based on the NS5A/NS5B site led to poor turnover of
the substrate by the HCV protease (Steinkühler et al., 1996b; Urbani et al., 1997),
indicating that the S1' pocket can accommodate residues much larger than the
natural Ser residue. In addition, a favorable interaction was found between the
P4' residue (Tyr) of these inhibitors and the NS4A cofactor (Landro et al., 1997).

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Incorporation of the optimized P'-side sequence and an N-terminal carboxylic acid,

which is well-positioned in the active site to engage in interactions similar to those
previously described for the C-terminal carboxylic acid of non-covalent product-
based inhibitors, resulted in a novel series of HCV NS3-4A protease inhibitors that
bind exclusively to the P'-side, without any contact with the P-side of the enzyme
(Ingallinella et al., 2002). Competitive, capped tripeptide inhibitors of the NS3-
4A protease, with low µM potency, were identified with the addition of a proper
linkage between these two elements from a small combinational library. Another
series of reversible, competitive inhibitors binding to the substrate-binding cleft
across the active site has been described (Colarusso et al., 2003). The presence of a
C-terminal phenethylamide group in these inhibitors allows an interaction with the
S'-side of the enzyme, which might present a potential alternative to the C-terminal
free carboxylic group present in the non-covalent, product analogue inhibitors.

IRREVERSIBLE INHIBITORS
As described above, the vast majority of HCV NS3-4A protease inhibitors that have
been described to date are either non-covalent or covalent but reversible inhibitors.
Recently, a series based on a pyrrolidine-5,5-trans-lactam core was reported to
inhibit the HCV protease with an IC50 up to 0.30 µM in replicon cell assay. These
inhibitors bind irreversibly to the enzyme through opening of the lactam ring, with
a biochemical potency (kobs/I) of 7,760 M-1s-1 for the best compound in this series
(Andrews et al., 2003a; Andrews et al., 2002; Andrews et al., 2003b; Andrews et
al., 2003c; Slater et al., 2002).

INTERACTION BETWEEN NS3 AND NS4A

Because the interaction with the NS4A co-factor is critical for maintenance of a
stable, active conformation of the NS3 protease, it is thought that agents competing
against the NS4A binding could be used to inhibit the HCV protease activity. A 14-
mer NS4A peptide (residues 21–34) with a substitution of Arg28 with Glu yielded
an IC50 of 20 µM against activation of the NS3 protease by the wild-type NS4A
peptide (Shimizu et al., 1996). 13-mer NS4A analogs (residues 22–34) assembled
from D-amino acids (instead of the normal L-amino acids) in a standard order, or
from L-amino acids in a reverse order, inhibited the NS3 protease activity with an
IC50 of 0.2 µM (Butkiewicz et al., 1996; Walker, 1999). In addition, several bivalent
inhibitors in which the above-mentioned NS4A analogs were fused in frame to a
NS5A/NS5B substrate have been described with IC50 values ranging from 0.2 µM
to 3 µM. In these cases, a hexapeptide (Glu-Asp-Val-Val-Cys-Cys), corresponding
to a P6–P1 portion of the NS5A/NS5B substrate, was fused via a short linker of
1–3 amino acids to the NS4A analogs (Walker, 1999). However, it is unclear how
these NS4A analogs inhibit the NS3-4A protease activity. Furthermore, since
formation of the NS3-NS4A non-covalent complex may occur co-translationally
during synthesis of the HCV polyprotein, it remains to be seen whether these NS4A

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analogs will be able to effectively compete against an already formed, tightly bound
NS3-NS4A complex.

APTAMERS
Another strategy to inhibit the HCV NS3-4A protease is to select aptamers (Biroccio
et al., 2002; Fukuda et al., 1997; Fukuda et al., 2000; Sekiya et al., 2003; Urvil et
al., 1997), which are single-stranded nucleic acids binding into a specific pocket of
the target protein with high affinity and interfering with function(s) of the protein.
Aptamers can be identified via multiple rounds of selection and amplification from
a pool of random nucleic acids against any protein or small-molecule target. Two
of these aptamers inhibit the HCV NS3 serine protease with an excellent potency
(Ki = 3 µM for the better aptamer inhibitor) (Kumar et al., 1997). In addition, the
same two aptamers were shown to block the helicase activity of NS3 (Kumar et al.,
1997). Inhibition of the NS3 protease activity by a different set of RNA aptamers
(Fukuda et al., 2000; Sekiya et al., 2003) was also demonstrated in transfected cells
(Nishikawa et al., 2003).

OTHER NON-PEPTIDIC SMALL-MOLECULE INHIBITORS

Several groups have undertaken high-throughput screening of large libraries of
chemical or natural products to identify novel inhibitors of the HCV NS3-4A
serine protease that are not peptidomimetic. Many of the confirmed hits are non-
competitive against the substrates (for a detailed review see Beaulieu and Llinas-
Brunet, 2002). One of the hit series is 2,4,6-trihydroxyl-3-nitrobenzamides (THNBs)
with an IC50 of 3.0 or 5.8 µM in the absence or presence of NS4A, respectively (Sudo
et al., 1997b). However, the major challenge for THNBs as potential therapeutic
agents against HCV is the lack of selectivity against human serine proteases,
such as chymotrypsin and elastase. Another series with a thiazolidine core was
identified by the same group of scientists, with an IC50 of 2.3 µg/mL (Sudo et al.,
1997a). Again, improvement in selectivity is the major challenge for this series
as the most selective derivative in this series showed a slight decline in potency.
In a recent structure-based NMR screening of a customized fragment library, 16
small-molecule hits were discovered to bind weakly, with a Kd in the range of 100
µM to 10 mM, to substrate binding sites of the HCV NS3-4A protease (Wyss et
al., 2004). NMR chemical shift perturbation data were then used to identify the
binding location and the orientation of the active site directed scaffolds. Two of
these compounds, which bind at the proximal S1–S3 and S2' substrate binding
pockets, were linked together to generate competitive inhibitors with relatively
high molecular weight and IC50 values in the µM range (Wyss et al., 2004). Novel,
Zn2+-dependent benzimidazole-based inhibitors of the HCV serine protease have
also been reported to form a ternary complex with a Zn2+ ion and the catalytic
residues, His57 and Ser195, as determined in X-ray crystallography. However, the
SAR for this series of compounds was established using a Zn2+-independent system,
and did not appear to be consistent in the presence of Zn2+ (Sperandio et al., 2002).

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The highly charged nature and the dependence on a high concentration of Zn2+
ion of this class of inhibitors present significant challenges in terms of cellular
penetration and oral bioavailability. Finally, a β-sheet mimetic has been combined
with the boronate ester warhead to create a potent series of inhibitors (Glunz et al.,
2003; Zhang et al., 2003).

PRECLINICAL STUDIES AND CLINICAL DEVELOPMENT

IN VITRO RESISTANCE MUTATIONS

Because of the error-prone nature of the viral reverse transcriptase of retroviruses
or RNA-dependent RNA polymerase of RNA viruses, drug resistance frequently
emerges in patients treated with antiviral drugs and therefore limits the efficacy of
these therapies. For new HCV NS3-4A serine protease inhibitors, resistance could
become a major issue in the treatment of patients. The HCV subgenomic replicon
system (Blight et al., 2000; Lohmann et al., 1999) was used for identification of in
vitro resistance mutations against two HCV protease inhibitor clinical candidates,
BILN 2061 or VX-950. All of the in vitro resistance mutations selected against
either inhibitor were substitutions of a single amino acid in the NS3 serine protease
domain and resulted in significant reduction in susceptibility to the respective
inhibitor (Lin et al., 2005a; Lin et al., 2004a; Lu et al., 2004). Two of the primary
resistance mutants against BILN 2061, Asp168-to-Val (D168V) and Asp168-to-Ala
(D168A), were highly resistant to BILN 2061 as reflected in at least a 63-fold
increase in Ki values in the FRET substrate-based enzyme assay and a more than
several hundred-fold jump in the IC50 values in the subgenomic replicon cell assay
(Lin et al., 2004a). However, both mutants at Asp168 remained fully susceptible
to VX-950 because there was only a slight decrease in both Ki and IC50 values in
enzyme and replicon cell assays, respectively (Lin et al., 2004a). Asp168 is in salt-
bridge interactions with the side-chains of Arg123 and Arg155, and is also part of
the S4 binding pocket. Computational modeling analysis suggests that substitution
of Asp168 with a non-acidic residue, such as Val or Ala, results in the loss of salt-
bridge interaction with the Arg155 side-chain on the neighboring β-strand (Fig. 5,
color coded in light green), which in turn makes multiple contacts with the large
P2 group of BILN 2061 in the model. Therefore, the conformation of the Arg155 in
the BILN 2061-wild-type NS3 protease complex is no longer energetically favored
in the D168V or D168A mutant. Instead, Arg155 in these two mutants appears to
clash with the P2 quinoline group of BILN 2061 and destabilizes its binding. On
the other hand, the conformation of Arg155 in the two published crystal structures
of the NS3 protease-inhibitor complex is similar to that in the VX-950-protease
complex (Fig. 5, color coded in orange). In addition, this conformation of Arg155
confers stabilization of VX-950 binding as it allows the maximal number of van
der Waals contacts between the Arg155 side-chain and the inhibitor. Therefore, VX-
950 is not expected to be affected by the substitutions at Asp168 compared with

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Fig. 5. Computational models of the HCV NS3-4A protease in complex with its inhibitors. The protein
is shown as a schematic based on its secondary structure in light gray. The inhibitors (VX-950 in
yellow and BILN 2061 in cyan) are shown as ball-and-stick models, with nitrogens in blue, oxygens
in red, and sulfur in orange. The side chains of Ala156 (green), Asp168 (orange) and Arg123 (orange)
are shown as sticks. The Arg155 side-chain of BILN 2061-protease model (R155-BI) is shown in light
green and that of VX-950-protease model (R155-VX) in orange. The catalytic triad, Ser139, His57, and
Asp81 is shown in gray. The figure was created using PyMOL Molecular Graphics Systems (DeLano
Scientific LLC, San Carlos, California).
A colour version of this figure is printed in the colour plate section at the back of this book.

BILN 2061 (Lin et al., 2004a). It should be noted that substitutions at Asp168 have
been identified in a previous study as the resistance mutations against a less potent
HCV protease inhibitor, which had an IC50 of about 1 µM in the replicon cell assay
(Trozzi et al., 2003). Another BILN 2061-resistant mutation, substitution of Arg155
with Gln (R155Q), was identified in a separate in vitro study. The R155Q mutant
was moderately resistant to BILN 2061 (a 24-fold increase in replicon cell IC50)
(Lu et al., 2004), although it is not clear whether this mutation confers resistance
to VX-950 or not.

The major in vitro resistance mutant against VX-950, Ala156-to-Ser (A156S), was
moderately resistant to VX-950 with a ~12-fold and ~29-fold increase in enzyme
Ki and replicon cellular IC50 values, respectively (Lin et al., 2004a). The HCV

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replicon cells containing the A156S substitution remained as sensitive to BILN

2061 as the wild-type replicon cells (Lin et al., 2004a). The Ala156 side-chain is in
van der Waals contact with the P2 group of these two inhibitors (Fig. 5, color coded
in green). In a computational model of the A156S mutant, the terminal oxygen of
Ser156 is too close to the P4 cyclohexyl group of VX-950, and it is also close to the
terminal cyclopentyl cap of BILN 2061. Because the cyclopentyl cap of BILN 2061
is at the flexible end of the inhibitor, it can be moved away from this unfavorable
contact without significantly affecting BILN 2061 binding. A similar movement
of the P4 cyclohexyl group of VX-950 causes destabilization of the interactions
between the inhibitor and S4 and S5 sub-sites of the protease. Therefore, a larger
loss in binding affinity is expected for VX-950 than for BILN 2061 with the A156S
mutant protease.

The lack of overlap between the dominant in vitro resistance mutations of BILN
2061 and VX-950 raised an interesting question – whether it is possible for a
combination of these two protease inhibitors to suppress the emergence of these
resistance mutations. While the combination did suppress the occurrence of A156S,
D168V or D168A mutants, two other single-residue substitutions, Ala156-to-Thr
(A156T) and Ala156-to-Val (A156V), were selected and found to confer cross-
resistance to both VX-950 and BILN 2061 (Lin et al., 2005a). In a computational
model, two out of the three possible conformations of the A156S side chain have
unfavorable contacts with both the inhibitors either at the P2 side chain or P3
carbonyl group. In the A156T or A156V mutation, the additional hydroxyl or methyl
group, respectively, at the Cβ atom of the residue 156 side-chain is forced to occupy
one of these two unfavorable positions, which leads to a repulsive interaction with
the inhibitor and/or enzyme backbone atoms. Therefore, A156T and A156V mutants
are expected to be resistant to both inhibitors (Lin et al., 2005a). It also remains to
be seen which of these resistance mutations identified in cell culture, if any, will
be observed in patients treated with HCV protease inhibitors.

IN VITRO COMBINATIONS
The current standard care for hepatitis C is a combination of weekly injections
of pegylated IFN-α and daily oral doses of ribavirin, which results in a SVR in
roughly half of treated patients. The SVR is higher (~80%) in patients infected
with genotype 2 or 3 HCV, but much lower (40-50%) in genotype 1 HCV-infected
patients, who account for the majority of hepatitis C population in developed
countries. It remains to be seen whether a single direct antiviral agent, such as
a protease inhibitor, is sufficient to induce a more favorable SVR in chronically
infected hepatitis C patients. One possible strategy to increase efficacy and help
suppress the emergence of resistance mutations in HCV protease inhibitor-based
therapy is to combine it with other antiviral agents, such as IFN-α or a polymerase
inhibitor. It has been shown that a combination of an HCV NS3-4A protease inhibitor

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with IFN-α resulted in a synergistic reduction of HCV RNA in the subgenomic

replicon cells after a 2-day incubation (Lin et al., 2004b). Furthermore, the benefit
of the combination was sustained over time such that a nearly 4-log10 or 10,000-
fold reduction of HCV RNA was achieved following a 9-day treatment of the HCV
replicon cells. The viral RNA dropped by more than 4-log10 to below the detection
limit after a-14 day combination treatment.

In the presence of G418 (neomycin), which allows selective growth of replication-

competent HCV replicon cells over "cured" Huh7 cells, no replicon cells were
recovered three weeks after withdrawal of the inhibitors, suggesting that the HCV
RNA has been cleared from the cells by the 14-day combination treatment (Lin et
al., 2004b). In each case, the antiviral effects obtained with higher concentrations of
either the protease inhibitor alone or IFN-α alone can be achieved by a combination
of both agents at lower concentrations, which potentially may reduce the risk of
possible adverse effects associated with high doses of either agent (Lin et al.,
2004b). Given the observation that HCV protease may interfere with the IFN signal
transduction pathway, one of the major components of the host anti-viral response,
these data suggest that HCV protease inhibitors may have a dual anti-HCV function,
blocking the HCV RNA replication and restoring the host antiviral response.

CLINICAL DEVELOPMENT
BILN 2061 (Fig. 4A, ciluprevir) was the first NS3-4A protease inhibitor to enter
clinical development. Despite its peptidic nature, BILN 2061 showed a low-to-
moderate oral bioavailability after a single dose in multiple species of animals,
including rats and dogs (Lamarre et al., 2003; Narjes et al., 2002b). In a single-
dose escalation phase 1a trial in healthy adults, this compound showed dose-
proportionality up to the 1,200 mg dose, and was well tolerated up to the 2,000 mg
dose. In several two-day, twice-daily dosing studies, BILN 2061 has been shown
to possess proof-of-concept antiviral activity in chronic genotype 1 HCV-infected
patients with minimal or advanced fibrosis (Benhamou et al., 2002; Hinrichsen et
al., 2002; Lamarre et al., 2003). At 200 mg/administration, a 2-3 log10 or greater
reduction in viral load was observed after the 2-day treatment. In some patients,
the viral load dropped below the limit of detection using a relatively insensitive
assay (<1500 copies/mL), although it remained positive in a more-sensitive assay
(>50 copies/mL). However, BILN 2061 was much less effective against genotypes
2 and 3 HCV, as evidenced by the Ki values (80–90 nM) against these proteases in
vitro (Thibeault et al., 2004), and an uneven, less pronounced viral load reduction
in the genotypes 2 and 3 HCV infected patients (Reiser et al., 2003). Unfortunately,
further development of BILN 2061 was put on hold due to safety concerns in
animals, which has not yet been fully disclosed.

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As described before, VX-950 (Fig. 4A) has a different mechanism of inhibition

than BILN 2061. VX-950 was the first covalent, reversible inhibitor of the HCV
NS3-4A protease to enter clinical development for hepatitis C. VX-950 showed
a moderate oral bioavailability and a much higher exposure in the livers than
in the plasma after a single dose in both rats and dogs (Perni et al., 2003b). In a
single-dose escalation (range 25–1,250 mg) phase 1a trial in healthy adults, VX-
950 was well tolerated up to the 1,250 mg dose level and exhibited good systemic
exposure, which was greater than proportional to dose (Chu et al., 2004). In a
recently completed 14-day study, VX-950 demonstrated excellent antiviral activity
in chronic genotype 1 HCV-infected patients (Reesink et al., 2005) (Fig. 6). Again,
VX-950 was well tolerated in all three groups, 450 mg thrice daily, 750 mg thrice
daily, and 1,250 mg twice daily. In all three groups treated with different VX-950
regimens, a 2–3 log10 or greater reduction in viral load was observed after the first
2 days of treatment, as in the case of BILN 2061. In some patients, the viral load
dropped by more than 4 log10 to below the limit of detection of a very sensitive
assay (<10 IU/mL) after 14 days of dosing. In addition, VX-950 was equally potent

Fig. 6. Antiviral antivity of VX-950 in chronic HCV-infected patients. Chronic genotype 1 HCV-
infected patients were treated with VX-950 at the following doses for 14 days: 450 mg thrice daily
(open square, n=10), 750 mg thrice daily (filled diamond, n=8), 1250 mg twice daily (filled triangle,
n=10), or placebo (open circle, n=6). The first dose was given at day 1, as indicated by a dashed line.
The plasma HCV RNA level was measured using COBAS Taqman HCV RNA assay, and the mean
plasma viral load of each treatment group is shown.

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against the HCV NS3-4A protease of genotype 2, but not genotype 3, in enzyme
assays (Taylor et al., 2004). A close homolog of VX-950 inhibited the replication
of a genotype 2a full-length HCV replicon that is capable of generating infectious
virus particle (Lindenbach et al., 2005), suggesting that it may also be an effective
agent for genotype 2 HCV-infected patients.

CONCLUSIONS AND FUTURE DIRECTIONS

The current standard therapy for chronic hepatitis C patients is a combination of
weekly injections of pegylated IFN-α, and daily oral doses of ribavirin. Both drugs
are indirect antiviral agents because they do not target a specific HCV protein or
nucleic acid. A SVR is achieved in only half of the treated patients and in less
than half of patients with genotype 1 HCV or with high viral load. The standard
therapy is associated with considerable adverse effects. There is a large unmet
medical need for orally available, small-molecule, direct anti-HCV drugs to provide
hepatitis C patients more effective treatments with fewer side effects. Ever since
the determination of HCV genome sequences in 1989 (Choo et al., 1989; Kuo et
al., 1989) and the identification and characterization of HCV proteins in the early
1990's, there have been intense efforts to discover novel direct antiviral drugs against
HCV. Determination of X-ray structures of the HCV NS3-4A serine protease and
the success of HIV protease inhibitors raised the hope of using structure-based
approaches to design a protease inhibitor against HCV. However, discovery of a
small-molecule, orally available, and potent drug candidate have been partially
hampered by the shallow substrate-binding groove of the HCV NS3-4A serine
protease. In addition, the lack of a robust small animal model for HCV infection
has generally forced scientists to rely on a combination of anti-HCV activity in
cell culture and animal pharmacokinetics as surrogate indicators of efficacy prior
to human trials. Nevertheless, significant progress has been made in recent years to
identify potent small-molecule inhibitors against the HCV protease. Clinical proof-
of-concept for HCV NS3-4A protease inhibitors has recently been obtained with
two inhibitors, BILN 2061 and VX-950. Given the lack of proof-reading function
of the HCV NS5B RNA-dependent RNA polymerase, potential drug resistance is
still a major concern for any direct antivirals against HCV, as in the case of HIV. It
remains to be seen whether HCV NS3-4A serine protease inhibitors will be used
as a monotherapy or in combination of other drugs, such as IFN-α, a polymerase
inhibitor, or both. Nevertheless, it will be very exciting to see HCV NS3-4A serine
protease inhibitors progress through clinical developments and, hopefully, provide
hepatitis C patients with much needed, more effective therapies.

ACKNOWLEDGMENTS
The author would like to thank Michael Briggs, Robert Kauffman, Steve Lyons,
Robert Perni, and John Thomson for the critical reading and editorial comments
on the manuscript.

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Chapter 7

HCV Helicase:
Structure, Function, and Inhibition
David N. Frick

ABSTRACT
The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3)
forms a three domain polypeptide that possesses the ability to travel along RNA or
single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled by ATP hydrolysis, this
movement allows the protein to displace complementary strands of DNA or RNA
and proteins bound to the nucleic acid. HCV helicase shares two domains common
to other motor proteins, one of which appears to rotate upon ATP binding. Several
models have been proposed to explain how this conformational change leads to
protein movement and RNA unwinding, but no model presently explains all existing
experimental data. Compounds recently reported to inhibit HCV helicase, which
include numerous small molecules, RNA aptamers and antibodies, will be useful
for elucidating the role of a helicase in positive-sense single-stranded RNA virus
replication and might serve as templates for the design of novel antiviral drugs.

INTRODUCTION
The C-terminal two thirds of the HCV NS3 protein forms an enzyme that is
seemingly unrelated to the serine protease discussed in the previous chapter. The
section of NS3 not needed for polyprotein cleavage folds into a three-domain
molecule that rapidly hydrolyzes all natural nucleoside triphosphates (NTPs)
and uses the resulting energy to move along a nucleic acid polymer dislodging a
complementary strand or bound proteins. All cells and many viruses express similar
proteins, which are called "helicases" because their biological role is normally to
unwind a DNA double helix. Since there is no DNA stage in the HCV lifecycle, the
exact function of HCV helicase is still unclear. It could unwind duplex RNA that is
formed when the single-stranded HCV genome is copied, or it might smooth RNA
secondary structures, which impede the NS5B RNA-dependent RNA polymerase.
Alternatively, the ability of HCV helicase to move like a motor along RNA could
be used for a process not linked to bona fide helicase activity (i.e. the disruption
of base pairs). HCV helicase could strip RNA binding proteins from viral RNA,
assist translation, or even help coordinate translation and polyprotein processing.
Regardless of its precise role in the HCV lifecycle, HCV helicase activity seems
necessary for viral replication, as evidenced by the fact that a mutated infectious

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clone, in which a point mutation abolishes the ability of NS3 to hydrolyze ATP,
does not replicate in chimpanzees (Kolykhalov et al., 2000).

In the last review devoted entirely to HCV helicase, Kwong et al. discussed its basic
biochemical properties, assay conditions, and the functions of conserved motifs
as revealed by the first crystal structures and initial structure-based site-directed
mutagenesis (Kwong et al., 2000). Several other reviews have also discussed some
of that material (Korolev et al., 1998; Yao and Weber, 1998; Frick, 2003; Frick,
2004). This chapter will therefore only briefly review many of these topics, and
will instead focus mainly on more recent mechanistic insights and HCV helicase
inhibitors that have been reported.

STRUCTURE OF HCV HELICASE

Unlike other systems where mechanistic experiments were carried out long before
protein-substrate interactions were viewed at an atomic resolution, the first crystal
structures of HCV helicase were solved only a few years after the protein was first
purified (Yao et al., 1997; Cho et al., 1998; Kim et al., 1998; Yao et al., 1999). These
structures are shown in Fig. 1. The helicase portion of NS3 forms three domains.
When viewed as a Y-shaped molecule, the most N-terminal domain (domain 1)
and the middle domain (domain 2) are above the C-terminal domain (domain 3).
In one structure (Kim et al., 1998), a short DNA oligonucleotide, containing only
the RNA base uracil, is bound to the helicase in the cleft that separates domain 3
from domains 1 and 2 (Fig. 1A). In several structures, a sulfate molecule is seen
bound between domains 1 and 2, in a position where ATP has been seen in high-
resolution structures of similar helicases (Soultanas et al., 1999; Velankar et al.,
1999; Bernstein et al., 2003) (Fig. 1A, C). When the entire NS3-NS4A complex is
viewed with the ATP and DNA binding sites in the front, the protease is in the back,
with its active site buried on the back of the helicase domains (Yao et al., 1999)
(Fig. 1C). Behind the protease is its NS4A cofactor, which positions the catalytic
triad of the protease so it will cleave the NS3-4A junction (Kim et al., 1996). The
zinc ion needed for the NS2-3 auto-catalytic protease lies on the same side of the
protease as NS4A (Fig. 1C). These crystal structures, along with high resolution
NMR structures of HCV helicase domain 2 (Liu et al., 2001; Liu et al., 2003), have
greatly influenced proposals explaining how helicases function and have guided
experiments designed to test these ideas.

Fig. 1. HCV Helicase Structures. A. PDB file 1A1V, showing the HCV helicase with a bound DNA
oligonucleotide and sulfate ion (Kim et al., 1998). The N-terminal RecA-like domain (domain 1) is
colored yellow, the C-terminal RecA-like domain (domain 2) is purple, and domain 3 is pink. DNA
and a sulfate ion (which occupies the ATP binding site) are depicted as spheres. (B) An electrostatic
surface of the protein in 1A1V calculated without the DNA using the program APBS (Baker et al.,
2001). Note the DNA is held in a negatively-charged pocket. (C) A full-length NS3 complex with the
central portion of NS4A covalently tethered to the NS3 N-terminus (Howe et al., 1999), as seen in

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PDB file 1CU1 (Yao et al., 1999). Helicase domains are colored as in panel A with the protease colored
green and NS4A blue. The protein is rotated about 90º relative to panel A. (D) An electrostatic surface
of the protein as viewed in panel C. Note that the positively-charged cleft surrounding the protease,
which could provide additional RNA binding sites. (E) Comparison of HCV helicase in the closed
conformation (PDB file 1HEI Yao et al., 1997) and the open conformation (PDB file 8OHM, Cho
et al., 1998). Proteins are superimposed along domains 1 and 3. (F) The model for a HCV helicase
dimer that was proposed by Cho et al. (1998). Protein domains are colored as in panels A and C. All
structures were rendered using the program Pymol (DeLano Scientific LLC, San Francisco, CA).
A colour version of this figure is printed in the colour plate section at the back of this book.

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STRUCTURAL VARIATIONS
Each of the available HCV helicase crystal structures is shown in one of the panels
of Fig. 1. Kim et al.'s structure (PDB accession code 1A1V, Kim et al., 1998) is
shown in Fig. 1 panels A and B, and Yao et al.'s structure (PDB 1CU1, Yao et al.,
1999) is in panels C and D. In panel E, Cho et al.'s structure (PDB 8OHM, Cho et
al., 1998) and Yao et al.'s structure (PDB 1HEI, Yao et al., 1997) are aligned for
comparison. Finally, Cho et al.'s model for a helicase dimer is shown in Fig. 1F.
The main difference between the available HCV structures concerns the position of
domain 2 relative to domains 1 and 3. Domains 1 and 3 share more of an interface
than domain 2 shares with either of the other domains. Domain 2 is connected to
domains 1 and 3 via flexible linkers, which allow domain 2 to freely rotate relative
to domains 1 and 3. In some structures, domain 2 is rotated away from domain 1 in
an "open" conformation, while in other structures domain 2 is closer to domain 1 in
a "closed" conformation (Fig. 1E). The pivot point for these rotations is provided
by additional contacts between domain 3 and an extended β-hairpin originating
from domain 2. An animation showing the rotation of domain 2 is available in the
Database of Macromolecular Movements (http://www.molmovdb.org/cgi-bin/
morph.cgi?ID=109065-518) (Echols et al., 2003).

How well these structures represent the diverse array of NS3 proteins encoded
by all varieties of HCV is not clear because natural variation in the amino acid
sequence of NS3 undoubtedly impacts its structure. The known HCV genotypes
have remarkably different nucleotide sequences, and the corresponding amino acid
substitutions likely would affect protein folding. HCV helicase from only three, very
similar, genotypes has been examined at the atomic level. Both Yao et al. (1997)
and Kim et al. (1998) examined an enzyme isolated from the same genotype 1a
H strain, Yao et al. (1999) examined the helicase from the genotype 1b BK strain,
and Cho et al. (1998) used an enzyme from another genotype 1b strain. Although
there are many differences between the structures, no obvious differences appear
to be genotype specific; the genotype 1a structures are as different from each other
as they are from the genotype 1b structures.

Nevertheless, variation in HCV helicase residues clearly influences its activity, as

evidenced by the fact that adaptive mutations in HCV replicons (see Chapter 11)
frequently arise in the helicase region (Blight et al., 2000; Krieger et al., 2001;
Grobler et al., 2003). To concisely depict helicase sequence variability among
various HCV genotypes, a consensus sequence of the NS3 peptide is superimposed
on a cartoon of the helicase structure in Fig. 2. This figure was generated by
rendering an alignment of all the NS3 sequences deposited in the hepatitis C virus
(HCV) database project (http://hcv.lanl.gov/) using a program called Weblogo
(http://weblogo.berkeley.edu/) (Crooks et al., 2004). The original alignment can
be downloaded from the HCV database by choosing "NS3" under the subheading

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Fig. 2. HCV NS3 sequence conservation. A sequence logo (Schneider and Stephens, 1990) of an NS3
sequence alignment is overlaid on a cartoon of the HCV helicase. Each residue of NS3 is depicted as
a stack of letters, the height of which correlates with how well it is conserved in 138 NS3 sequences
deposited in the HCV database (http://hcv.lanl.gov/). The height of the letters in each stack correlates
with how frequently that amino acid occurs at that position. Conserved superfamily 2 helicase motifs
and other key residues are noted and highlighted with bold type.

"alignments." Weblogo depicts an alignment as a sequence logo (Schneider and

Stephens, 1990), in which each NS3 residue is represented as a stack of one
letter amino acid codes. The height of each stack corresponds to the amino acid
conservation at that position. When the residue is invariant, only one letter is shown,
and the most common substitutions are noted when the residue is variable.

Lam et al. (2003b) have explored the impact of genotypic variation on the various
activities of HCV helicase by examining recombinant proteins that were isolated
from infectious clones of HCV genotype 1a (Yanagi et al., 1997), 1b (Yanagi
et al., 1998), and 2a (Yanagi et al., 1999). Although there are some differences
between the genotypes, the proteins are surprisingly similar. The main difference
between genotype 1 and 2 strains can be attributed to variation at residue 450,
which is normally a Thr (Fig. 2), but in the genotype 2a infectious clone it is an Ile

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(Yanagi et al., 1999). When Thr450 alone is changed to Ile, the protein binds ssDNA
differently and unwinds DNA faster, suggesting that the interaction of Thr450 with
DNA observed in the crystal structure somehow modulates the rate of helicase
movement. Upon close examination of sequences that were later deposited in the
HCV database, it appears now that only the particular genotype 2a strain used
in our study (Lam et al., 2003b) contains the Ile substitution. Since this was an
infectious clone isolated from a chimpanzee (Yanagi et al., 1999), we now believe
that T450I is an adaptive mutation that permits the virus to efficiently replicate in
chimpanzees, but it is not normally seen in HCV infecting humans.

CONSERVED MOTIFS
The sequence logo in Fig. 2 also reveals that there are numerous stretches of amino
acids that do not vary in known isolates. The numbered sequence motifs are shared
with related helicases (Gorbalenya and Koonin, 1993; Hall and Matson, 1999).
Some of these motifs, such as the DExD/H-box portion of motif II and motif IV
are characteristic only of helicases closely related to HCV helicase, while others,
such as the Walker A motif (Motif I), are conserved among all helicases and in a
wide variety of other proteins that hydrolyze ATP. Other motifs are found only in
HCV and closely related viruses, including the Arg-clamp, the Phe loop (Lam et
al., 2003a), and all motifs in domain 3.

It was not possible to precisely depict all the conserved residues in Fig. 2 relative to
their position within each domain of HCV helicase, but most are close. Diagrams
noting exact motif positions on actual crystal structures have been published
elsewhere (Hall and Matson, 1999; Kwong et al., 2000; Lam et al., 2003a; Frick,
2004). Motifs I, Ia, II, III, IV, V, and VI, which are conserved in similar helicases
encoded by both viruses and cellular organisms, line the ATP binding cleft, and
some of these motifs project residues into the nucleic acid binding site. These
seven helicase motifs essentially form the motor which converts the chemical
energy derived from ATP hydrolysis into a mechanical force that drives helicase
movements leading to the disruption of DNA or RNA base pairs.

The roles of most of the key conserved residues in motifs I through VI have been
investigated using site-directed mutagenesis. The various studies are tabulated in
Table 1 along with a phenotype of each mutant. The phenotype listed in Table 1
simply depicts whether helicase activity (i.e. DNA or RNA unwinding), ATPase, or
nucleic acid binding properties of each mutant is unchanged (normal), diminished,
or enhanced. Mutations in motifs I-VI normally impact the ability of the protein to
both unwind DNA and hydrolyze ATP, showing that the two activities are coupled.
However, sometimes mutations result in decreases of ATP hydrolysis rate, but
not a similar corresponding decrease in DNA unwinding. The results have been
interpreted by some authors as evidence that ATP hydrolysis is not absolutely

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required for unwinding, but they could instead be due to the different sensitivities
of ATPase and helicase assays under the particular conditions utilized.

Outside motifs I to VI, domains 1 and 2 have regions that are both variable and
conserved. With a hope of discovering regions that might provide binding sites for
novel anti-HCV therapeutics, our lab examined the role of two motifs in domain
2 that are conserved in all HCV isolates but not related proteins. The rationale
was that compounds that bind such sites would be relatively non-toxic because
similar sites are not present on related cellular helicases. The first motif identified
centered on Arg393, a residue that contacts the nucleic acid backbone. When Arg393
is changed to Ala, the protein still catalyzes RNA-stimulated ATP hydrolysis but
does not unwind DNA or RNA. The R393A protein also binds DNA weaker both
in the presence and absence of a non-hydrolyzable ATP analog, suggesting that this
Arg-clamp motif functions to tether the protein to the nucleic acid strand on which
it is translocating (Lam et al., 2003a).

The second motif characteristic of only helicases from strains of HCV and related
viruses forms a loop connecting two β-sheets that extend from domain 2. The β-loop
structure is composed of residues Thr430 to Ala452, and a pair of residues, Phe438
and Phe444 and is located in a highly conserved region at the loop's tip. The turn
of the loop is composed of NS3 amino acids 438 to 444. At the time, the function
of this "Phe-loop" was a curiosity. Kim et al. (1998) had proposed that this loop
functions like a DNA binding loop found in ssDNA binding proteins. Alternately,
Yao et al. (1997) proposed that Phe438 and Phe444 could pack into a hydrophobic
pocket together with Phe531, Phe536, and Trp532, allowing the loop to take on a more
structural role. Both Phe438 and Phe444 were altered to Ala to assess these two very
different possibilities. Mutagenesis of the Phe's that flank the Phe-loop demonstrates
that the loop is not involved in nucleic acid binding. Rather, Phe438 and Phe444
are important both for proper protein folding and for modulating conformational
changes leading to the release of DNA upon ATP binding (Lam et al., 2003a).

All helicases crystallized to date contain domains that resemble domains 1 and 2,
but none share a domain that resembles domain 3. In some helicases, such as PcrA
(Subramanya et al., 1996) and Rep (Korolev et al., 1997), two domains replace
domain 3, one which extends from domain 1 (called domain 1B) and one that extends
from domain 2 (called domain 2B). In several helicase structures that share domains
similar to domains 1 and 2 of HCV helicase, such as the RecQ protein (Bernstein
et al., 2003), DnaG (Singleton et al., 2001), and eukaryotic translation initiation
factor 4A (Caruthers et al., 2000), domain 3 is missing entirely, suggesting that
domain 3 might not be required for HCV helicase movements. This is not the case,
however, and although its role in unwinding is only beginning to be understood,
domain 3 is clearly essential. Deletion of 97 amino acids from the C-terminus of

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Table 1. HCV helicase mutants.

Mutant Motif Phenotype Reference(s)
A204V I An, B+, H- Tai et al., 2001
K210A I A-, Bn, H- Heilek and Peterson, 1997; Levin and Patel, 1999; Min et
al., 1999; Wardell et al., 1999
K210N I A-, B+, H- Tai et al., 2001
K210Q I A-, H- Heilek and Peterson, 1997
K210E I A-, Bn, H- Kim et al., 1997b; Chang et al., 2000
S231A Ia A+, B+, Hn Lin and Kim, 1999
T266A H-, No dimer Khu et al., 2001
Y267S H-, No dimer Khu et al., 2001
T269A TxGx A-, B-, H- Lin and Kim, 1999
D290A II A-, Bn, H- Levin and Patel, 1999; Min et al., 1999; Wardell et al.,
1999
- n -
D290N II A,B ,H Tai et al., 2001
E291A II A-, Bn, H- Wardell et al., 1999; Tai et al., 2001
E291Q II A-, Bn, H- Tai et al., 2001
C292G II A-, Bn, Hn Kim et al., 1997b
C292S II A-, Bn, H- Kim et al., 1997b; Wardell et al., 1999
C292A II A-, Bn, H- Tai et al., 2001
M288T H-, No dimer Khu et al., 2001
H293A II A+, B+, H- Heilek and Peterson, 1997; Kim et al., 1997b; Tai et al.,
2001
H293K II A-, B+, H- Tai et al., 2001
H293Q II A-, B+, H- Tai et al., 2001
T322A III A-, B+, H- Kim et al., 1997b; Tai et al., 2001
T324A III A-, Bn, H- Tai et al., 2001
n, Bn, Hn
H369A IV A Frick et al., 2004a
H369K IV A+, B+, H- Frick et al., 2004a
S370A IV An, Bn, Hn Lin and Kim, 1999
Y392A An, B-, H- Paolini et al., 2000
R393A Arg-clamp An, B-, H- Lam et al., 2003a
T411A V A+, B-, H- Lin and Kim, 1999
V432A A-, Bn, H- Paolini et al., 2000; Preugschat et al., 2000; Tai et al.,
2001
V432D A-, B-, Hn Kim et al., 2003
V432R A+, B+, Hn Kim et al., 2003
F438A Phe-loop An, B+, H- Lam et al., 2003a
F444A Phe-loop A-, B-, H- Lam et al., 2003a
T450I An, B+, H+ Lam et al., 2003b
-, Bn, H-
Q460A VI A Kwong et al., 2000
Q460H VI A-, B+, H- Kim et al., 1997b; Wardell et al., 1999
R461A VI A-, Bn, H- Kim et al., 1997b; Kwong et al., 2000
R461Q VI A-, B-, H- Tai et al., 2001
R462A VI A+, Bn, Hn Kwong et al., 2000

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Table 1. Continued.
R462L VI A-, B-, H- Kim et al., 1997b; Chang et al., 2000
G463A VI A-, Bn, Hn Kim et al., 1997b
R464A VI A-, Bn, H- Kim et al., 1997b; Min et al., 1999; Chang et al., 2000;
Kwong et al., 2000
T465N VI A-, Bn, Hn Kim et al., 1997b
G466A VI A-, Bn, H- Kim et al., 1997b
R467A VI A-, Bn, H- Kwong et al., 2000
K VI A-, Bn, H- Kim et al., 1997b; Wardell et al., 1999
E493K A+, B+, H+ Frick et al., 2004a
E493Q A+, B+, H+ Frick et al., 2004a
W501A An, B-, H- Lin and Kim, 1999; Paolini et al., 2000; Preugschat et al.,
2000; Tai et al., 2001; Kim et al., 2003
W501L An, B-, H- Lin and Kim, 1999
W501F An, Bn, Hn Lin and Kim, 1999; Preugschat et al., 2000; Kim et al.,
2003
W501E An, B-, H- Kim et al., 2003
W501R An, B-, H- Kim et al., 2003
n Normal ATPase
A
Hn Normal duplex unwinding
A+ Enhanced ATPase
H+ Enhanced duplex unwinding
A- Poor ATPase
H- Poor duplex unwinding
Bn normal nucleic acid binding
B+ Enhanced nucleic acid binding
B- Poor nucleic acid binding

NS3 results in an inactive helicase (Jin and Peterson, 1995; Kim et al., 1997a). Two
key residues in domain 3 are Trp501, which stacks against a nucleic acid base to act
like a bookend (Lin and Kim, 1999; Preugschat et al., 2000; Kim et al., 2003), and
Glu493, which helps repel nucleic acids from the binding cleft upon ATP binding
(Frick et al., 2004a).

MECHANISM OF ACTION
There is presently no consensus on exactly how the HCV helicase unwinds RNA.
Debate about the HCV helicase mechanism continues largely because in some
experiments, HCV helicase appears to function as a monomer, but in others it
appears to be a dimer or a higher order oligomer. Below, attempts will be made to
reconcile this and some other controversies, but first, to understand the intricacies
of these molecular models, it will be necessary to review a few fundamental
characteristics of all helicases.

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BASIC PROPERTIES OF ALL HELICASES

All helicases can be divided into two basic groups. Some form rings that encircle
DNA (or RNA) while others, like HCV helicase, do not form rings. Both ring and
non-ring helicases, are primarily associated with one strand of a double helix and
can be classified based on the polarity of that strand. The protein either shifts from
the 3'-end to the 5'-end or from the 5'-end to the 3'-end on the strand to which it is
mainly bound. The most common method to diagnose the direction of movement
is to determine whether the helicase requires a 5'-ssDNA tail or a 3'-ssDNA tail to
initiate unwinding. 5'-3' helicases need a 5'-ssDNA tail, and 3'-5' helicases require
a 3'-ssDNA tail. HCV helicase is a 3'-5' helicase (Tai et al., 1996; Morris et al.,
2002). As a consequence, if the oligonucleotide bound to HCV helicase in PDB file
1A1V (Fig. 1A) (Kim et al., 1998) represents the strand on which HCV helicase
translocates, then the duplex portion of the helix would likely be positioned to
the right of the protein in Fig. 1A (see cartoon in Fig. 2). Helicases are thirdly
classified based on their evolutionary relationships. Gorbalenya and Koonin have
used protein sequence comparisons to classify most helicase families into one of
three large superfamilies (Gorbalenya and Koonin, 1993). Non-ring helicases are
generally members of helicase superfamily 1 (SF1) or superfamily 2 (SF2), while
ring helicases are in superfamily 3 (SF3) or in other families not in the three main
superfamilies. HCV helicase is a member of SF2, and like all helicases in SF2,
shares conserved motifs I through VI described above.

The ring formed by ring helicases usually is composed of six identical subunits
assembled in a head-to-tail manner. The rings surround the strand on which the
helicase is translocating and the complementary strand passes outside the ring
(Egelman et al., 1995). ATP binds between the subunits, to the head of one subunit
and the tail of an adjacent protomer. There are consequently six ATP binding sites
per hexameric ring (Singleton et al., 2000). Each subunit of a ring helicase contains
a single domain that resembles a domain first seen in the structure of a protein called
RecA, which plays a key role in E. coli DNA recombination (Story and Steitz,
1992). In ring helicases, ATP hydrolysis leads to rotation of the RecA-like domains
which in turn leads to movements of positively-charged loops that protrude into the
center of the ring. The positively charged loops bind DNA (Notarnicola et al., 1995;
Washington et al., 1996), and the sequential interaction of the DNA-binding loops
with DNA is thought to lead to ring helicase movement (Singleton et al., 2000).

In non-ring helicases, like HCV, there are two RecA-like domains in a single protein
subunit, and ATP binds between these subunits. In HCV helicase, domains 1 and 2
fold into similar structures although they share no apparent sequence homology. The
core of both domains is composed of a series of beta sheets sandwiched between
sets of alpha helices. Both domains 1 and 2 are similar to RecA, form the ATP
binding site, and contact DNA. The main structural difference between domains 1

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and 2 is that domain 2 contains two long beta sheets that project towards domain
3 (the Phe-loop discussed above), which are not present in domain 1.

MECHANISM OF ATP HYDROLYSIS

Although the position of ATP bound to HCV helicase has not yet been visualized,
the mechanism of its hydrolysis most likely resembles that seen in other helicases.
The approximate configuration of ATP in the binding site can be seen by comparing
a HCV helicase structure with one of a similar helicase that has been crystallized
in the presence of a non-hydrolyzable ATP analog. Fig. 3A shows the results of
a structural alignment of HCV helicase (PDB file 1A1V) with the SF2 helicase
RecQ bound to ATPγS (PDB file 1OYY) (Bernstein et al., 2003). Shown only are
the ATPγS (from 1OYY), the HCV helicase, and its bound oligonucleotide (both
from 1A1V). Residues that likely play key roles in ATP hydrolysis are highlighted
as sticks.

The configuration of residues at the ATP-binding site depicted in Fig. 3A is

reminiscent of that seen in all other helicases that have been studied bound to NTPs
(Sawaya et al., 1999; Soultanas et al., 1999; Velankar et al., 1999; Singleton et al.,
2000; Bernstein et al., 2003; Gai et al., 2004; James et al., 2004). ATP and a required
metal ion cofactor (depicted as Mg2+ in Fig. 3A) normally bind to a helicase in the
cleft that separates two adjacent RecA-like domains. The most critical residues
for ATP binding arise from the Walker A and B motifs (Walker et al., 1982). The
Walker A motif of HCV helicase forms a phosphate binding loop (P-loop) with
the conserved Lys210 likely contacting the γ phosphate of ATP. The Walker B motif
contains acidic residues that coordinate the positively charged divalent metal
cation, which in turn contacts the phosphates of ATP. In the alignment in Fig. 3A,
Asp290 seems to be ideally suited to coordinate the catalytic metal. In or near the
Walker B motif of helicases and related proteins, there is normally a residue which
acts as a catalytic base by accepting a proton from the water molecule that attacks
the γ phosphate of ATP. Normally, the catalytic base in this class of enzymes is a
glutamate (Goetzinger and Rao, 2003; Orelle et al., 2003), and Glu291 seems to be
properly positioned to perform this function.

A more detailed analysis of HCV structures suggests that the roles of particular
residues might be somewhat more complicated than assumed above. For example,
to function as a catalytic base, the pKa of Glu291 would need to be much higher than
that of a typical Glu in a protein. However, electrostatic analysis of all HCV helicase
structures reveals that neither Glu291, nor any nearby Glu, has an abnormally high
pKa. In contrast, Asp290 has a pKa as high as 10 in some structures and as low as
3 in others. Interestingly, in structures in the open conformation (such as 8OHM),
the pKa of Asp290 is low, and in the closed conformation (ex. 1A1V), the pKa of
Asp290 is higher than 7, suggesting that Asp290 picks up a proton (like a catalytic

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Fig. 3. Key residues in HCV helicase. (A) HCV helicase residues likely involved in modulating ATP
binding and hydrolysis. The approximate position of ATP bound to HCV helicase is revealed by a
structural alignment of HCV helicase (PDB file 1A1V) (Kim et al., 1998) with the SF2 helicase
RecQ bound to ATPγS (PDB file 1OYY) (Bernstein et al., 2003) (B) Key residues contacting the
oligonucleotide bound to HCV helicase in PDB file 1A1V (Kim et al., 1998).

base) when the protein changes from the open to the closed conformation. Thus,
Asp290 could serve as a catalytic base instead of, or in addition to, coordinating the
magnesium-ATP complex (Frick et al., 2004a).

Once the water molecule is activated, it acts as a nucleophile, most likely attacking
the terminal phosphate of ATP. The pentavalent transition state, where the γ
phosphate is bound to 5 oxygen atoms, then breaks down into ADP and inorganic
phosphate. In similar ATP hydrolyzing enzymes, the transition state is stabilized by
one or more positively charged residues, normally arginines, that function in concert
with the lysine of the Walker A motif. In many enzymes, including helicases, these
"arginine fingers" are frequently part of adjacent protein subunits. For example, in

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 HCV Helicase

small GTPases like Ras, the Arg-finger that activates GTP hydrolysis is part of the
GTPase-Activating Protein (GAP) (Ahmadian et al., 1997). In F1ATPase, an Arg-
finger on the alpha subunit stabilizes the transition state (Nadanaciva et al., 1999).
Recently, it was shown that in ring helicases, the Arg-finger and P-loop are part of
different polypeptide chains in the hexamer (Crampton et al., 2004), demonstrating
why ring helicases need to oligomerize to cleave ATP. In HCV helicase, several
arginines are present in domain 2, and they line the ATP binding cleft. These residues
are part of conserved motif IV and include Arg461, Arg462, Arg464, and Arg467. In the
model shown in Fig. 3, Arg467 and Arg464 are nearest the phosphates of ATP. Either
could rotate even closer to ATP if the cleft between domains 1 and 2 closes. Of the
two, only Arg467 is shown because it is of the most interest. Arg467 is methylated by
cellular protein arginine-methyltransferase I (Rho et al., 2001). Although it is still
unclear how methylation influences helicase activity, such a modification should
eliminate all activity if Arg467 acts as an Arg-finger. Site-directed mutagenesis
supports this contention. When Arg467 is changed to Lys (Kim et al., 1997b; Wardell
et al., 1999) or Ala (Kwong et al., 2000), the proteins do not unwind RNA, and
ATPase activity is decreased over 10-fold. An R464A mutant has a similar effect
(Kim et al., 1997b; Min et al., 1999; Kwong et al., 2000).

Many of the other conserved residues help to properly position the above groups by
forming networks of hydrogen bonds, ionic bonds, and hydrophobic interactions.
In addition, some conserved residues help coordinate the rotation of domain 2. Two
such amino acids are noted on Fig. 3A. His293 in motif II (domain 1) and Gln460
in motif VI (domain 2) are near each other in many structures and could interact.
Kim et al. called these residues "gatekeepers" and propose that they might provide
a switch modulating the opening and closure of the cleft between domains 1 and 2
upon ATP binding (Kim et al., 1998). Mutation of either residue has a profound and
interesting effect on ATPase. Mutation of Gln460 abolishes detectable ATPase, but
an H293A mutation results in a protein with a significantly higher level of ATPase
in the absence of RNA, and the protein still unwinds RNA. In the presence of
RNA, the H293A mutant hydrolyzes ATP slower than wildtype, to such an extent
that RNA appears to inhibit ATP hydrolysis (Kim et al., 1997b). These data further
support the idea that rotation of domain 2 is related to ATP hydrolysis, and that
domain closure leads to a completion of the active site and hydrolysis of ATP. The
question of how ATP hydrolysis is translated into helicase movement on nucleic
acid still remains unanswered, however. The first two models that were applied to
HCV helicase to explain its movements suggest that either the protein operates as
a monomer like an inchworm (Kim et al., 1998) or as a dimer, which rolls along
nucleic acid (Cho et al., 1998).

THE INCHWORM AND ROLLING MODELS

If ATP binding and hydrolysis leads to movement of domain 2 relative to domain 1,
then this conformational change would in turn impact interactions between residues

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in these two domains with RNA. A close-up of the DNA binding site of structure
1A1V is shown in Fig. 3B, with key amino acids highlighted. Unlike SF1 helicases,
which have many interactions with nucleic acid bases (Velankar et al., 1999), most
of the contacts occur with protein side chains and the sugar-phosphate backbone of
DNA (Kim et al., 1998). One key hydrogen bond is donated from domain 1 residue
Thr269, which is part of the conserved TxGx motif, and an analogous interaction
arises from motif V residue Thr411 in domain 2. Mutagenesis of either residue
affects both RNA binding affinity and unwinding rates (Lin and Kim, 1999). Also
noted on Fig. 3B are residues Thr450, Arg393, Trp501, and Glu493, whose roles were
alluded to above.

Unlike ring helicases that need to oligomerize to cleave ATP because the Arg-finger
is located on the opposite side of a protein monomer relative to the Walker A motif,
all the residues necessary for ATP hydrolysis are present in a single polypeptide
chain in HCV helicase. Monomeric models for HCV helicase action state that upon
rotation, ATP binding leads to a closure of the cleft between domains 1 and 2 by a
rotation of domain 2 relative to the rest of the protein, a movement first observed in
the structures of Yao et al. (1997). Such conformational changes conceivably could
cause the protein to act like an inchworm to move along RNA. Most monomeric
models are variations on the "ratcheting inchworm" model first proposed for HCV
helicase by Kim et al. (1998). Based on the observation that the oligonucleotide
appears to be locked into the binding cleft because a residue in domain 3, Trp501, is
stacked against the 3'-terminal base, Kim et al. proposed that ATP binding, and the
subsequent closure of the cleft between domains 1 and 2, will lead to a ratcheting
of Trp501 past 1 or 2 nucleotides. Consequently, the protein would move towards
the 5'-end of the bound nucleic acid. After ATP is hydrolyzed and Trp501 is again
locked into place acting as a bookend, the cleft opens and RNA slides through
the other side of the protein. Kim et al. proposed that the residue that acts as a 5'-
bookend, analogous to the 3'-bookend Trp501, might be Val432 in domain 2 (Kim
et al., 1998).

The dimeric models for HCV helicase action are essentially variations on Wong
and Lohman's rolling dimer hypothesis that was used to explain the actions of a
dimer formed by the E. coli Rep helicase upon DNA binding (Wong and Lohman,
1992). In the rolling dimer, each subunit alternates between a form that prefers to
bind ssDNA and a form that preferentially binds a double helix. Switching between
the states is modulated by ATP binding and hydrolysis. In theory, both forms are
bound to a DNA fork, with one subunit bound to the ssDNA tail, and the other
bound to the duplex region. When the trailing subunit changes conformation so
that it prefers to bind duplex DNA, it will roll toward the double helix causing the
subunit bound to the duplex to wrench one strand away from its complement so
that it can then bind the resulting ssDNA (for review see Lohman and Bjornson,

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 HCV Helicase

1996). A modified rolling model was applied to the HCV helicase by Cho et al.
(1998), who observed that two HCV helicase monomers could pack in the manner
shown in Fig. 1F. Cho et al. called their model a "descending molecular see-saw"
and proposed that RNA could thread through a long cleft formed between domains
1 and 2 of adjacent subunits (Cho et al., 1998). However, the later structure by Kim
et al. (1998) showing DNA bound in another cleft (Fig. 1A), coupled with the fact
that ATP likely binds in the cleft between domains 1 and 2 (Fig. 3), makes such an
orientation seem unlikely.

EVIDENCE FOR A FUNCTIONAL MONOMER (THE INCHWORM MODEL)

Ever since the HCV helicase portion of NS3 was first purified, it was apparent that
it behaved as a monomer and did not need to oligomerize to cleave ATP. Initial
studies found HCV helicase to act as a monomer in solution based on gel filtration
(Preugschat et al., 1996) and analytical ultracentrifugation (Porter et al., 1998). As
discussed above, the monomeric enzyme has all the residues necessary to catalyze
ATP hydrolysis, and as a result, no decrease in turnover number (kcat) is observed
when HCV helicase is diluted (Levin and Patel, 2002). In contrast, diluting a ring
helicase leads to a loss of the ability to hydrolyze ATP at low protein concentrations
(Guo et al., 1999). There is also yet no direct structural evidence for dimerization.
Although the protein exists as a dimer in the crystallographic asymmetric unit in
PDB files 1HEI (Yao et al., 1997) and 1CU1 (Yao et al., 1999), it is a monomer
in 1A1V (Kim et al., 1998) and 8OHM (Cho et al., 1998). No evidence has been
presented that interfaces seen in PDB files 1CU1 or 1HEI are biologically relevant,
and Cho's model (Fig. 1F) is based on crystal packing interactions, not actual
observed interfaces (Cho et al., 1998).

Soon after the ratcheting inchworm model was introduced for HCV helicase (Kim
et al., 1998), it was tested by several groups using site-directed mutagenesis (Table
1). Most initial interest focused on the residues that act as bookends, or the teeth
of the ratchet. Several groups have confirmed the importance of Trp501 in both
nucleic acid binding and unwinding (Lin and Kim, 1999; Paolini et al., 2000;
Preugschat et al., 2000; Kim et al., 2003). Without a bulky aromatic amino acid at
position 501, HCV helicase is unable to unwind RNA (Lin and Kim, 1999; Tai et
al., 2001; Kim et al., 2003) but retains some ability to unwind DNA (Kim et al.,
2003), albeit more slowly than wild type (Preugschat et al., 2000). The data is less
clear regarding the residue that might bookend the 5'-end of the RNA. Kim et al.
propose that this residue is Val432 in Domain 2 (Kim et al., 1998), but Paolini et
al. have suggested that Tyr392 could play a similar role (Paolini et al., 2000). Some
of these reports have suggested that mutation of these residues leads to decreases
in helicase activity (Paolini et al., 2000; Preugschat et al., 2000; Tai et al., 2001;
Kim et al., 2003).

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More recently, other predictions made by the inchworm model have been tested.
One basic prediction is that binding of ssDNA to the cleft separating domain 3
from domains 1 and 2 activates ATP hydrolysis. Supporting this theory, several
mutations have been made in the DNA binding cleft that decrease the affinity of
the protein for both DNA and RNA and affect rates of ATP hydrolysis (see Table
1) (Kim et al., 1997b; Lin and Kim, 1999; Tai et al., 2001). However, there exists
an alternate explanation for such results. The mutant proteins might not fold
properly or may be less stable than the wildtype, explaining the decreased binding
and unwinding activity. In contrast to these negative results, our lab has recently
found that substitution of one of two residues in the DNA binding cleft, His369 or
Glu493, enhances binding and lowers the amount of nucleic acid needed to stimulate
ATP hydrolysis. For example, an E493K mutant enhances binding to RNA in the
presence of ATP by several orders of magnitude. This positive result provides the
clearest evidence that DNA/RNA binding to this region activates ATP hydrolysis
(Frick et al., 2004a).

Another prediction made by the inchworm model is that the ssDNA bound between
domain 3 and domains 1 and 2 is the strand on which the helicase is translocating
in a 3' to 5' direction. We have tested this idea by analyzing a helicase in which
Arg393 is changed to Ala. Without this Arg-clamp in the DNA binding cleft (Fig.
3B), the protein cannot unwind DNA or displace proteins bound to ssDNA. The
R393A protein retains full RNA-stimulated ATPase activity, and still binds ssDNA
with the same stoichiometry as wildtype, albeit more weakly. These data provide
strong evidence that the protein moves along the strand seen in the crystal structure
in a 3' to 5' direction and the duplex region would lie as diagramed in Fig. 2 (Lam
et al., 2003a).

The inchworm model lastly predicts that ATP binding should modulate the affinity
of the protein for RNA (or ssDNA). Early kinetic studies of nucleic acid stimulation
of ATP hydrolysis suggest that, indeed, ATP binding weakens the affinity of the
protein for RNA (Preugschat et al., 1996). However, it was difficult to confirm this
observation by directly measuring dissociation constants because HCV helicase only
weakly interacts with most common non-hydrolyzable ATP analogs. A breakthrough
came when Levin et al. found that the presence of BeF3 tightly locks the reaction
product, ADP, on the enzyme (Levin et al., 2003). In other systems, BeF3 coordinates
like the γ phosphate of ATP, so that ADP(BeF3) is essentially a non-hydrolyzable
ATP analog (Xu et al., 1997). Using ADP(BeF3), Levin et al. showed that when
ATP binds HCV helicase, affinity for DNA falls by almost two orders of magnitude.
More recently, Lam et al. have shown that nucleic acid binding to HCV helicase is
pH dependent in the presence of ADP(BeF3) but not in the absence of the analog,
demonstrating that a conformational change occurs upon ATP binding (Lam et al.,
2004), as is also predicted by the inchworm model.

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EVIDENCE FOR A FUNCTIONAL OLIGOMER (THE ROLLING MODEL)

While the evidence that HCV helicase acts as a monomer is convincing, there is
also evidence that multiple subunits interact with each other to efficiently unwind
RNA. Yeast two-hybrid assays provide the most persuasive evidence that NS3
interacts with itself (Flajolet et al., 2000; Khu et al., 2001). In such experiments,
the minimum peptide required for an NS3-NS3 interaction contains only domain
1 residues 162-335. This peptide surrounds conserved motifs I, II, and III (Khu et
al., 2001), suggesting domain 1 would interact with domain 1 of another monomer,
rather than domain 1 interacting with domain 2 as proposed by Cho et al. (1998) (Fig.
1F). Three residues, which were identified using a reverse two hybrid screen, are
critical for dimer formation, Thr266, Tyr267 and Met288 (Khu et al., 2001). Met288 is
not conserved and is normally an Ile in all but a few HCV isolates (Fig. 2). Mutations
of these residues not only influence dimer formation that can be assayed using gel
filtration, but also the ability of the protein to unwind DNA (Khu et al., 2001).

Oligomerization of NS3 seems to be dependent on nucleic acids. Before the yeast

two-hybrid data were reported, Levin and Patel demonstrated that DNA aids the
ability to chemically crosslink HCV helicase into high molecular weight species
(Levin and Patel, 1999). Dimerization of NS3 has also been visualized using
analytical gel filtration, but only in the presence of an oligonucleotide (Khu et al.,
2001). Nucleic acid binding data can sometimes be fit to models that do not take
into account subunit interactions (Porter, 1998b; Porter, 1998a; Porter et al., 1998;
Levin and Patel, 2002), but under certain conditions, cooperative models fit the
data better (Locatelli et al., 2002; Frick et al., 2004b). Taken together, these data
suggest that two or more HCV helicase protomers cooperatively assemble onto
ssDNA (or RNA) in a controlled manner.

The latest evidence for oligomerization has emerged from measurements of rates of
HCV helicase catalyzed DNA and RNA unwinding. Notably, unwinding rates are
not linearly dependent on the amount of protein present in the reaction, but rather,
accelerate greatly once a critical protein concentration is reached (Lam et al., 2003a;
Frick et al., 2004b). By measuring unwinding under single-turnover conditions,
several groups have presented kinetic models explaining this cooperativity (Levin
et al., 2004; Serebrov and Pyle, 2004; Tackett et al., 2005). As reviewed elsewhere
(Bianco, 2004), these models all take into account the interaction of multiple
protomers aligned on a ssDNA (or RNA) strand and attempt to calculate the number
of base pairs unwound in a single turnover event (called "step size"). The theory
holds that an oligomer would unwind many base pairs (10 or more) in a single
event while a monomer would only unwind a few base pairs at a time. Levin et al.
(2004) have calculated a step size of 9 base pairs using DNA, and using a long RNA
substrate, Serebrov and Pyle have determined that 18 base pairs are unwound by
HCV helicase in a single step (Serebrov and Pyle, 2004). These values support a

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rolling dimer model and stand in stark contrast with an older calculation by Porter
et al. that only a few base pairs of fluorescently-labeled DNA are unwound by HCV
helicase in a single turnover event (Porter et al., 1998).

OTHER MECHANISMS EXPLAINING HELICASE MOVEMENT

Because neither the inchworm nor the rolling model fully explains all elements of
helicase action, additional models have been recently proposed. One such model
states that HCV helicase acts like a Brownian motor (Astumian, 1997, Levin et al.,
2005 #1004). A Brownian motor exploits random movements that constantly occur
on the molecular level (Brownian motion) and an asymmetrical path to shift an object
in a single direction. As diagrammed in Fig. 4A, collisions that occur between HCV
helicase, water, and other small molecules constantly transfer small amounts of
momentum to the protein so that it wobbles slightly relative to the RNA to which it
is bound. In the absence of ATP, the helicase is constrained in a certain location due
to molecular barriers. However, when HCV helicase binds ATP, it releases its grip
on RNA by changing conformation so that it is free to move along RNA. Random
collisions will then be more likely to transfer enough momentum that the protein
clears the barrier constraining it to its original position. The key to the Brownian
motor model is the asymmetry of the path on which the motor is traveling. Because
the path is asymmetrical in the Brownian motor model, the protein will be more likely
to move in one direction than the other, and the net result of many movements will
be movement in a single direction. If the path were symmetrical, then the protein
would be equally likely to return to the original position as it would be to move to
either adjacent position, and the net result would be no movement.

In Fig. 4, this irregularity is depicted as an asymmetrical free energy diagram. If

the helicase moves in a 5' direction, then it will likely clear a free energy peak and
descend to the base of another valley towards the 5' end of the RNA. On the other
hand, if the protein moves toward the 3' end of the RNA by the same amount, it will
not clear the peak and will return to the position at which it began the cycle. Thus,
even though movements occur randomly in either direction and many molecules will
remain in the same location, the net result will be that most molecules will move in
the same 3' to 5' direction. This model has been applied to HCV helicase by Levin
et al. (2003), who recently observed that HCV helicase has a higher affinity for a
partially duplex DNA substrate with 3'-ssDNA tails, than it does for either ssDNA
alone, or DNA with a 5'-ssDNA tail. They propose that interaction with the fork of
the DNA leads to asymmetry of the free energy diagram (Levin et al., 2005). While
movements towards the fork likely play some role in helicase movement, the fact
remains that two groups have independently observed translocation of HCV helicase
in a 3' to 5' direction in systems containing no duplex portion on the ssDNA substrate
(Morris et al., 2002; Lam et al., 2003a). Thus, if the Brownian model holds true for
HCV helicase, then all asymmetry in the free energy diagram should be intrinsic
to the helicase and the nucleic acid strand on which it is traveling.

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Fig. 4. Two possible mechanisms for HCV helicase translocation on RNA. (A) The Brownian motor
model (Levin et al., 2005). In the absence of ATP, HCV helicase is confined in a single location on
an asymmetrical path of RNA. When ATP binds, binding releases the protein from RNA, allowing
random movement (Brownian motion) to transport the helicase either in a 5' or 3' direction. Because
the path is asymmetrical, molecules moving in the 3' direction will return to their original position,
whereas molecules moving in the 5' direction will change positions. Net movement will be in a 5'
direction. (B) The propulsion-by-repulsion model (Frick et al., 2004a; Lam et al., 2004). ATP binding
rotates domain 2 so that a positively charged Arg-clamp (Lam et al., 2003a) moves the RNA so that
it clears Trp501, which is holding the RNA in a negatively charged cleft. When ATP is bound, the
protein repels RNA past Trp501 so that the protein moves in a 5' direction until ATP is hydrolyzed and
the protein returns to its original conformation.

Our lab has proposed another model to explain HCV helicase movement that
suggests that HCV helicase utilizes electrostatic forces to move along DNA and
RNA (Frick et al., 2004a; Lam et al., 2004). This "propulsion-by-repulsion" model
(Fig. 4B) is based on two observations. First, DNA is tightly bound in a pocket

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of the enzyme that is highly negatively charged (see Fig. 1B). Second, release of
DNA from the enzyme is pH dependent; the enzyme binds weaker to DNA in the
presence of ATP at a higher pH. The first observation hints that there is a potential
energy buildup when the protein is locked onto DNA in the absence of ATP. The
second observation suggests that ionizable residues come in contact with DNA upon
ATP binding. We have shown using mutagenesis that one of these key residues is
Glu493 in the ssDNA binding cleft (Frick et al., 2004a). In our model, ATP binding
leads to a conformational change such that the nucleic acid bases can clear the
Trp501 bookend (Lam et al., 2004). In the absence of ATP, RNA cannot exit the
enzyme because it is blocked by Trp501 and clamped in the cleft by the Arg-clamp
on domain 2 (Lam et al., 2003a). When ATP binds, domain 2 rotates bringing
with it the positively-charged Arg-clamp. The Arg-clamp attracts the negatively-
charged phosphodiester backbone so that RNA moves free from the bookend. The
negatively-charged RNA is then repelled by the negatively charged binding cleft,
so it moves through the protein until ATP is hydrolyzed, and the protein clamps it
tightly again. In such a model, the step size of the helicase would depend on the
nature of the nucleic acid on which the protein is translocating explaining, in part,
why different step sizes have been calculated using different substrates (Porter et
al., 1998; Levin et al., 2004; Serebrov and Pyle, 2004).

ROLE OF THE PROTEASE DOMAIN AND NS4A

Reviewing the HCV helicase literature is perplexing because frequently the data
reported in one study differs from that reported in other studies. Rates of ATP
hydrolysis, DNA/RNA unwinding, and dissociation constants frequently differ by
more than 10-fold. The most likely explanation for such differences (when the same
protocol is used) is that most labs utilize different recombinant versions of HCV
helicase. Many of these proteins are quite different because they either (1) include
different portions of NS3, or (2) have been isolated from different HCV strains.
Because full-length NS3 is difficult to express in E. coli, a truncated protein containing
only NS3 residues 166-631 is frequently used. However, some studies have used
helicase constructs with more or fewer N-terminal NS3 residues. Furthermore, some
studies use a helicase lacking fusion proteins, whereas other studies utilize a helicase
with a N-terminal or C-terminal His-tags, a T7-tag, a GST-tag, or combinations of
multiple tags. Frequently, the tags are not removed before analysis.

We have compared numerous NS3 constructs in our laboratory to try to understand

why different studies have reported such different results. Initially, we thought that
such variation might be due to intrinsic differences between the HCV genotypes.
Our comparison of three helicases isolated using the same procedure from three
different genotypes noted some differences, but these tended to be small (less than
2-fold) and did not explain the widely divergent data in the literature (Lam et al.,
2003b). We then set out to compare the effect of fusion proteins, which are attached

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to the helicase to aid expression and purification, and found that these modifications
led to major changes in activity (Frick et al., 2004b). While modifications to the C-
terminus did not affect most assays, modification to the N-terminus did, suggesting
that the protease domain and the conformation of the region linking it to the helicase
could have a major role in aiding the cooperative assembly of the protein on RNA
and in unwinding (Frick et al., 2004b).

In our hands (Frick et al., 2004b), full-length NS3 (with NS4A) unwinds RNA better
than versions lacking the protease, but hydrolyzes ATP slower, suggesting that it
is a more efficient molecular motor. Some of the effects of the protease could be
substituted for by GST or His-tag fusion proteins, but several could not, suggesting
that RNA makes specific contacts with the protease region. As discussed above, it
is not clear where the complementary strand or the duplex region of RNA interacts
with HCV helicase. Nevertheless, an electrostatic analysis of the full-length protein
(Fig. 1D) reveals that a positively-charged cleft is formed between the protease
and domain 2 of the helicase. Residues in this cleft could tether the protein to the
negatively-charged phosphate backbone of RNA. It is possible that a similar cleft
could be formed when the protease is replaced with a fusion protein, explaining
why such proteins have a higher apparent processivity than the helicase domain
alone (Frick et al., 2004b).

Not all other studies have noted as clear differences when the protease domain
is removed from NS3. For example, even though Kuang et al. found that a NS3-
NS4A complex unwinds RNA better than an isolated helicase domain, they also
noted NS3 lacking NS4A is a poor helicase relative to an isolated helicase domain,
suggesting that the protease without its NS4A cofactor might actually inhibit helicase
movements (Kuang et al., 2004). Similarly perplexing data showing relatively
poor helicase activity for full-length NS3 have been reported by others (Heilek
and Peterson, 1997; Gallinari et al., 1998). The poor helicase activity of some
full-length NS3 constructs could be explained by the conformational flexibility
of the protein. In order to cleave the rest of the polyprotein, Yao et al. proposed
that the protease domain swings away from the helicase via the flexible linker that
connects the two regions (Yao et al., 1999). If this occurs, then the putative RNA
binding cleft proposed above would be disrupted and the helicase would more
rapidly dissociate from RNA substrates.

A model in which RNA binds a cleft between domain 2 of the helicase and the
protease would also provide a plausible role for the NS4A peptide in facilitating
helicase action. NS4A could hold the protein in a conformation so that the RNA
binding cleft between the protease and helicase remains intact, explaining why
some investigators find that a NS3-NS4A complex unwinds RNA better than NS3
alone. For example, Pang et al. (2002) compared the activities of a NS3-NS4A

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complex expressed in insect cells (Sali et al., 1998) with a His-tagged, full-length
NS3 protein expressed and purified from E. coli, and found that the NS3-NS4A
complex requires less time to form a functional complex on RNA. Based on the
structure of the NS3-NS4A complex (Fig. 1C), it is difficult to envision a direct
interaction between NS4A and RNA, as has been proposed by others (Silverman
et al., 2003). Thus, we prefer a model where NS4A stabilizes the formation of an
RNA binding cleft on NS3 (Frick et al., 2004b).

In addition to being a better RNA helicase, we also find that the full-length
protein oligomerizes more readily than the truncated protein lacking a protease
domain, indicating that the protease domain properly configures the protein for
oligomerization. As evidence, we find twice as many protomers of full-length NS3
bound to a single oligonucleotide as recombinant proteins containing the helicase
domain only (Frick et al., 2004b). This key observation explains why early studies
using isolated helicase domains lacking the protease failed to detect cooperative
assembly (Preugschat et al., 1996; Levin and Patel, 2002; Lam et al., 2003a), while
later studies using the full-length NS3 often detect oligomers (Khu et al., 2001;
Locatelli et al., 2002; Frick et al., 2004b).

HCV HELICASE INHIBITORS

Because compounds that inhibit a helicase encoded by herpes simplex virus (HSV)
have been recently shown to moderate disease symptoms (Crute et al., 2002;
Kleymann et al., 2002), there has been great interest in finding inhibitors of HCV
helicase. Many compounds that inhibit HCV helicase have been reported, and they
can be broadly classified as small molecules, nucleic acids, or antibodies. There
are, however, many obstacles that must be overcome before developing helicase
inhibitors into viable antiviral agents. The main problem will likely be toxicity
because the motor domains of HCV helicase are conserved in a vast array of cellular
proteins. Consequently, there is more focus on finding inhibitors that bind sites that
are not conserved with cellular enzymes, such as the RNA binding site(s) described
above and possible allosteric regulatory sites. Even if these inhibitors are never
developed into drugs, they should still be useful for elucidating the role of HCV
helicase in the viral lifecycle.

SMALL MOLECULES
Many of the small molecules that were initially examined as HCV helicase inhibitors
were nucleoside analogs. Although nucleoside analogs might also inhibit cellular
proteins by interacting with conserved Walker sequences, there is some potential
for these compounds because there is a possibility that nucleotides could bind to a
second site on the helicase in addition to the conserved Walker site. Such a site could
be formed, for example, if the protein oligomerizes and ATP binds to an interface
between the RecA-like domains of adjacent subunits. Porter et al. first detected a

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possible second nucleotide binding site when they studied product inhibition in
the presence of NaF. In their studies, about two moles of ADP bound per protein
monomer (Porter, 1998a). In contrast, as discussed above, when beryllium fluoride
is added to the reaction, only one mole of ADP is bound per protomer (Lam et al.,
2003a; Levin et al., 2003). One model explaining these data assumes that the helicase
functions as a dimer with ATP bound tightly to the interface between domains 1
and 2 and ADP bound more weakly to a second interface. ADP fluoride complexes
likely do not resemble the substrate, ATP, as closely as ADP(BeF3), so they might
bind both active and allosteric sites. Also in support of a second NTP-binding site,
Locatelli et al. have demonstrated that nucleotides bind HCV helicase cooperatively
(Locatelli et al., 2002).

There is also some evidence that the second potential nucleotide binding site on
HCV helicase is more specific than the nucleotide binding site between domains
1 and 2. The alignment in Fig. 3 reveals few contacts are made between HCV
helicase and the sugar or base of an NTP, explaining the observed non-specificity
of this site. HCV helicase hydrolyzes all eight canonical nucleoside triphosphates
(Preugschat et al., 1996; Wardell et al., 1999; Lam et al., 2003b). The seven other
(d)NTPs are competitive inhibitors of ATP hydrolysis (Lam et al., 2003b) and
most studies find that they all support unwinding. However, one study found that
only some NTPs fuel unwinding with efficiency comparable to that seen with ATP
(Locatelli et al., 2001). Other (d)NTPs, particularly dATP, were found to be poor
substrates and potent inhibitors of unwinding (Locatelli et al., 2001). Such results
can be explained if NTP binding to a regulatory site is more specific than NTP
binding to the catalytic site. For example, the regulatory site might only bind dATP
but not the NTPs that fail to inhibit unwinding.

Regardless of whether nucleoside analogs will ever be developed into anti-

HCV therapeutics, the effects of such compounds on HCV helicase have been
extensively studied. Examples include ribavirin triphosphate (Borowski et al.,
2001), 5'-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine
(FSBA), guanosine (FSBG) and inosine (FSBI) (Bretner et al., 2004), and ring-
expanded ("fat") nucleosides and nucleotides (Zhang et al., 2003). Much of the data
has been previously reviewed (Borowski et al., 2002a; Borowski et al., 2002b).
Generally, such compounds inhibit only at very low ATP concentrations, and are
competitive with ATP, so that under physiological conditions little or no inhibition
is observed.

Compounds resembling nucleoside bases have also been reported to be

HCV helicase inhibitors. For example, tetrachlorobenzotriazole (TCBT) and
tetrabromobenzotriazole (TBBT) were recently analyzed as helicase inhibitors. Both
compounds inhibit unwinding catalyzed by helicases from related viruses (such as

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West Nile virus) with IC50's in the low micromolar range, but only TBBT inhibits
RNA unwinding by HCV helicase (IC50 ~60 μM). Neither compound inhibits
helicase-catalyzed ATP hydrolysis (Borowski et al., 2003), but it is still not clear
whether these compounds bind a true allosteric site or if they inhibit unwinding by
non-specific interactions with the nucleic acid substrate.

Many groups have reported non-nucleoside based inhibitors of HCV helicase,

primarily in the patent literature. These compounds include a piperidine derivative,
heterocyclic carboxamide, antracycline antibiotics (Borowski et al., 2002b),
paclitaxel, trifluoperazine (Borowski et al., 2002a), and aminophenylbenzimidazole
derivatives (Phoon et al., 2001). Many of these compounds intercalate in nucleic
acids and likely act via that non-specific mechanism. Whether or not any of these
compounds or other small molecules decrease HCV replication measured using
replicons or animal models is still yet to be reported.

NUCLEIC ACID BASED INHIBITORS

One of the unique properties of HCV helicase is that, unlike other helicases, the
protein binds RNA and DNA in a sequence specific manner. Even the first studies of
the protein noted that HCV helicase has a distinctive nucleic acid stimulation profile
(Suzich et al., 1993). This means that ATP hydrolysis is stimulated by some nucleic
acid polymers much better than it is stimulated by others. The range is quite dramatic.
Poly(G) RNA does not stimulate at any measurable level, and poly(U) RNA (or DNA)
stimulates best (up to 50-fold). Interestingly, differential stimulation is not entirely
due to differences in binding affinity. Direct binding assays confirm that poly(U)
binds HCV helicase tighter than polymers composed of the other bases (Gwack et
al., 1996), but at saturating nucleic acid concentrations, not all sequences support the
same maximum rate of ATP hydrolysis, suggesting that the protein assumes different
conformations when bound to different sequences (Lam et al., 2003b).

Previously, I have proposed that HCV helicase nucleic acid specificity stems from
interactions between conserved amino acids in the RNA binding cleft, particularly
Trp501, Glu493, and Asn556, with nucleic acid bases, either directly or through water
molecules (Frick, 2004). As evidence supporting this hypothesis, mutation of Trp501
has been reported to result in a protein that is more efficiently stimulated by poly(C)
than poly(U) RNA (Lin and Kim, 1999), and there is a change in the nucleic acid
stimulation profile when Glu493 is substituted by another amino acid (D. N. Frick and
R. S. Rypma, unpublished results). RNA specificity has also been proposed to play
a role in directing the helicase, and possibly the entire HCV replication complex,
to certain regions of the viral genome. For example, HCV helicase specifically
binds both to the 3'-UTR and the 3'-end of the negative strand viral transcript (the
complement of the 5'-UTR). This might be necessary during the viral lifecycle to
allow the NS5B polymerase to synthesize RNA in these regions that contain stable
secondary structures (Banerjee and Dasgupta, 2001).

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Nucleic acid interactions with HCV helicase depend not only on the base composition
but also on the composition of the nucleic acid backbone. It is widely recognized
that HCV helicase unwinds a DNA duplex more efficiently than an RNA duplex.
The biological reason for this, if there is one, is still a mystery because there is no
DNA stage in the viral lifecycle, and replication likely occurs on the endoplasmic
reticulum (Wolk et al., 2000). However, some reports have detected NS3 in the
nucleus (Muramatsu et al., 1997; Errington et al., 1999), where the helicase could
modify host gene expression (Sakamuro et al., 1995). Whereas loss of the 2'-OH
group from RNA permits the helicase to unwind substrates faster (DNA is unwound
faster than RNA), adding a methyl group to this position (2'-O-methyl RNA)
weakens helicase interaction with RNA and prevents unwinding (Hesson et al.,
2000). The effects are strand specific in that the helicase only appears to sense the
chemical composition of the strand with the 3'-overhang (the longer strand in the
helicase substrate). Composition of the shorter strand does not affect unwinding rates
as drastically, suggesting that interactions are made primarily with the nucleic acid
sugars of only one strand of the helicase substrate. When the longer strand (with
the 3' overhang) is DNA, the shorter strand can be composed of RNA, 2'-O-methyl
RNA, morpholino-DNA, or phosphorothioate-DNA without affecting unwinding
(Hesson et al., 2000; Tackett et al., 2001; Pang et al., 2002). However, if the shorter
strand is composed of peptide nucleic acid (where a N-(2-aminoethyl)glycine
backbone replaces the deoxyribose phosphates), then unwinding is slower than with
natural substrates (Tackett et al., 2001). Whether peptide nucleic acids are poor
substrates because of the lack of specific interactions with HCV helicase, or simply
because they form more stable duplexes, is still unclear (Tackett et al., 2001).

Two groups have tried to exploit the nucleic acid specificity of HCV helicase
with a goal of developing RNA-based inhibitors. Both groups have used SELEX
(systematic evolution of ligands by exponential amplification) to find RNA aptamers
that tightly bind HCV helicase. In the SELEX procedure, an RNA library is screened
for sequences that bind a macromolecule. Only those sequences that bind tightly
are amplified to create a new library, and the selection process is repeated with the
new library. Although directly using RNA as an antiviral drug will be challenging
because of its cellular instability, the information derived from aptamer studies could
be used to make more stable derivatives or by delivering RNA directly to infected
cells using gene therapy (for more on anti-HCV nucleic acids see Chapter 18).

One set of aptamers specific to HCV helicase was generated by modifying aptamers
that bind tightly and inhibit the NS3-NS4A serine protease. Such aptamers were
found to bind truncated NS3 lacking the helicase domains using SELEX. They
all share the conserved sequence GA(A/U)UGGGAC (Fukuda et al., 2000), bind
NS3 protease over one thousand times tighter than random RNA sequences and

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are effective at inhibiting the HCV NS3 protease (Fukuda et al., 2000; Nishikawa
et al., 2003). When positions Arg130, Arg161, and Lys165 are substituted with Ala,
the aptamers no longer bind NS3, suggesting that they interact near these NS3
residues, which are located in the region that links the protease to the helicase
(Hwang et al., 2000). To create an aptamer that inhibits protease and helicase
activity of NS3, a 14-mer uridine tail was added to one of the most effective HCV
protease-binding RNA aptamers. The new, longer aptamer interacts with both the
protease and helicase domains of the full-length NS3 protein (Fukuda et al., 2004),
binds to the helicase portion of NS3 with high affinity (Kd ~4 nM) (Fukuda et al.,
2004), and inhibits the NS3 helicase activity with an EC50 of ~500 nM. The same
group has recently reported a new "advanced dual functional" aptamer in which
another aptamer, selected for helicase binding (Nishikawa et al., 2004), is tethered
to a protease-binding aptamer using a poly(U) linker. This new aptamer is about
five times more effective than either aptamer when they are not covalently linked
(Umehara et al., 2005).

A second group has also selected for aptamers using the helicase portion of NS3
as the bait in the SELEX procedure (Hwang et al., 2004). This aptamer (called
SE RNA) folds to form four stem loops with GC pairs that are similar to the stem
loop located at the 3'-terminal of the negative strand HCV RNA. This observation
suggests that the SE aptamer might bind the helicase in a similar manner as the
stem loop located at the 3'-terminal of the negative strand HCV RNA (Banerjee and
Dasgupta, 2001). SE RNA binds the HCV helicase tightly (Kd ~990 pM), efficiently
competes with poly(U), stimulates ATP hydrolysis, and potently inhibits RNA
unwinding (IC50 ~12.5 nM). When delivered to human liver cells (Huh 7) infected
with HCV replicons, the SE aptamer slows HCV RNA synthesis, and interestingly,
labeled SE aptamers can also be used as a diagnostic tool to detect the NS3 protein
in cells from HCV patients (Nishikawa et al., 2004).

ANTIBODIES
The third, and possibly most ambitious, method that is currently being explored to
inhibit HCV helicase is to generate antibody-like molecules that, when expressed
intracellularly, will bind and inhibit HCV helicase activities. Almost all HCV
patients produce antibodies directed against the NS3 protein, and the vast majority of
these bind to the helicase portion of the protein (Chen et al., 1998). Several groups
are working toward the goal of introducing recombinant antibodies into cells for
"cellular immunization," a procedure which has been used experimentally with HIV
(Goncalves et al., 2002). In this approach, HCV infected cells are transfected with
a gene expressing a portion of an antibody selected for reactivity with NS3. One
method is to use single chain fragment (ScFv) antibodies. A ScFv is composed of
the immunoglobulin heavy chain variable domain connected to the variable region
of the light chain by a polypeptide linker. Such a molecule can be constructed using

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PCR. The other principal method uses an antibody fragment (Fab), which contains
the complete light chain and the variable and first constant domains of the heavy
chain. A Fab is larger and usually more stable than a ScFv.

To construct a ScFv, immunoglobulin specific PCR is first used to construct a library

of human antibody fragments using plasma cells from HCV patients as the PCR
template. To identify which antibodies react with HCV helicase, the fragments are
fused to a bacteriophage coat protein for phage display, and phages with a high
affinity for HCV helicase are purified. Tessman et al. have used this technique to
isolate a series of high affinity ScFv's that specifically interact with HCV helicase
(Tessmann et al., 2002). ScFv's that bind HCV helicase have also been constructed
by splicing together the variable domains of monoclonal antibodies (Zhang et al.,
2000; Sullivan et al., 2002), and after expression and purification, several of these
recombinant proteins inhibit HCV helicase-catalyzed DNA unwinding (Sullivan
et al., 2002; Artsaenko et al., 2003). One particular ScFv consists of the variable
regions of the human monoclonal antibody CM3.B6, which recognizes an epitope
that spans conserved SF2 helicase motifs IV and V (Mondelli et al., 1994). The
CM3.B6 ScFv has been expressed in HCV infected hepatocytes (HepG2 cells),
immunoblots of which reveal an intra-cellular interaction between the antibody
and NS3. HCV RNA synthesis within primary hepatocytes infected with HCV is
also reduced by 10-fold when the cells contain a vector carrying the CM3.B6 ScFv
gene (Sullivan et al., 2002).

Phage display has also been used to isolate an anti-HCV helicase Fab from a patient
infected with HCV genotype 1b. Prabhu et al. have isolated this human Fab, called
HFab-aNS3, and demonstrated that it has HCV antiviral activity (Prabhu et al.,
2004). HFab-aNS3 recognizes an epitope that spans motifs I to V of the protein,
and when purified and pre-incubated with HCV helicase, HFab-aNS3 abolishes
detectable DNA unwinding. Intracellular expression of HFab-aNS3 within replicon-
transfected Huh 7 cells suppresses NS3 protein expression and significantly inhibits
viral RNA synthesis of both subgenomic and full-length HCV replicons (Prabhu
et al., 2004).

CONCLUSIONS AND FUTURE DIRECTIONS

HCV helicase has attracted the attention not only of researchers interested in
developing novel antiviral drugs, but also those studying how proteins interact
with nucleic acids. As one of only three helicases that have been crystallized bound
to an oligonucleotide, HCV helicase has become one of the best model proteins
to study how helicases unwind duplexes and move on DNA and RNA. Different
theories on how chemical energy stored in ATP is transformed into the mechanical
force necessary to move a protein from one position to another are currently hotly
debated, and many have been tested using the HCV NS3 protein. Obviously, work
will continue until HCV helicase's precise mechanism of action is defined.

233
Frick

While it is still uncertain if HCV helicase functions using an inchworm, rolling,

Brownian, or electrostatic mechanism, extensive work has uncovered its basic
properties and the roles of several key residues. ATP binds HCV helicase between
two RecA-like domains, causing a conformational change that leads to a decrease
in the affinity of the protein for nucleic acids. Key residues contacting ATP include
Lys210, which likely coordinates the phosphates, Asp290, which could coordinate
a divalent metal ion, Glu291, which might act as a catalytic base, and one or more
arginines on the adjacent domain. One strand of RNA binds in a second cleft
formed perpendicular to the ATP-binding cleft and its binding leads to stimulation
of ATP hydrolysis. RNA and/or ATP binding likely causes rotation of domain 2 of
the enzyme relative to domains 1 and 3, and somehow this conformational change
allows the protein to move like a motor. Key residues involved in RNA binding
include Trp501, which locks the protein in position in the absence of ATP, and Glu493,
which repels RNA when ATP binds.

Additional structural and mechanistic work may or may not be necessary in order
to develop helicase inhibitors into antiviral drugs. For example, much less is
known about the structure and mechanism of action of the herpes simplex virus
helicase that is the target of newly developed antiviral drugs (Kleymann, 2004).
In contrast, the biological role of the HSV enzyme in viral replication is much
more clearly defined than that of the HCV helicase. The HSV helicase targeted
by antiviral drugs is needed to coordinate RNA primer synthesis on the lagging
strand while the double helix is unwound (Boehmer and Lehman, 1997). All that
is firmly established about the biological function of HCV helicase is that if its
ability to hydrolyze ATP is abolished, the virus will no longer infect chimpanzees
(Kolykhalov et al., 2000).

Clearly, the biological role of HCV helicase needs to be investigated in more detail.
Perhaps the many inhibitors or site-directed mutants of HCV helicase could be
used to design experiments elucidating its role using the replicon system. If the
helicase is only needed to unwind duplex RNA formed after RNA synthesis, then
replicons with a defective helicase should still synthesize small amounts of both
the polyprotein and negative sense RNA. However, when an antibody against
HCV helicase is co-expressed in cells expressing HCV replicons, there is not only
diminished positive strand synthesis but also less synthesis of HCV negative strand
RNA and HCV proteins, suggesting that the helicase plays numerous complex and
important roles in the viral lifecycle (Prabhu et al., 2004). Given the propensity for
HCV helicase to unwind DNA, issues regarding possible roles of the NS3 protein
in manipulating cellular DNA should also be more thoroughly examined.

While presently it appears that HCV protease and HCV polymerase inhibitors will
be developed as the next generation of anti-HCV drugs, compounds inhibiting

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HCV helicase might also someday prove therapeutically useful. Both NS5B and
NS3 protease have clearer roles in HCV replication, and unlike helicases, the
mechanisms of serine proteases and RNA polymerases have been understood for
decades. Consequently, it is not surprising that HCV helicase inhibitor development
lags behind that for the other HCV enzymes. As long as its mechanism and role
in replication are not clearly understood, development of antiviral drugs targeting
HCV helicase will remain difficult. Nevertheless, rapid progress is being made in
the helicase field, and it will not be surprising if HCV helicase inhibitors someday
enter clinical trials.

ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health grant AI052395. I am
grateful to Angela M. I. Lam and Ryan S. Rypma for reviewing the manuscript and
Christopher M. Frenz for help preparing the figures.

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Chapter 8

HCV NS4B:
From Obscurity to Central Stage
Ella H. Sklan and Jeffrey S. Glenn

ABSTRACT
The hepatitis C virus (HCV) non-structural 4B (NS4B) protein is a 27kDa
hydrophobic protein which for many years was characterized mainly as a protein
of unknown function. Recently, however, information about the protein and
its involvement in mediating various viral activities and effects on host cells is
beginning to accumulate. NS4B has been implicated in modulation of NS5B's RNA
dependent RNA polymerase activity and various host signal transduction pathways,
a possible role in HCV carcinogenesis, impairment of ER function, and regulation
of both viral and host translation. Perhaps most significant, NS4B has recently
been found to be responsible for the formation of a novel intracellular membrane
structure, termed the membranous web, which appears to be the platform upon
which viral replication occurs. Specific domains within NS4B have been identified
which likely underlie the mechanisms employed by NS4B to mediate many of the
preceding functions. As such, these domains which include an amphipathic helix and
nucleotide-binding motif represent attractive targets for new antiviral strategies.

INTRODUCTION
With the cloning of HCV (Choo et al., 1990) it was possible to deduce many of the
expected protein products encoded in the viral genome. Among these was a 261
aa protein now known as NS4B. Unlike several other predicted protein products
of the HCV polyprotein, no obvious functions could be immediately ascribed
to NS4B. For years, NS4B essentially remained a membrane-associated protein
characterized mainly by a lack of known function. Recently, however, considerable
information concerning NS4B as begun to accumulate. These efforts have both led
to a realization of NS4B's importance to the viral life cycle and yielded attractive
new targets for antiviral drug development. This chapter will attempt to review these
developments and speculate on some of the future directions in this increasingly
exciting field. After discussing NS4B genesis, localization and topology, NS4B's
relationship with a specialized type of membrane will be addressed. A survey of
various NS4B properties and functions will follow, including NS4B's role in the
induction of a novel type of membrane structure which represents the candidate site
for HCV replication. Finally, we will focus on features within NS4B which may
underlie the mechanisms employed by NS4B to mediate its associated functions.

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PROTEOLYTIC GENERATION OF NS4B

The HCV genome is translated from a single ~3000 amino acid-long open reading
frame into a large polyprotein that is processed both co- and posttranslationally by a
combination of host and viral proteases (see Fig 1). Cleavages generating the HCV
structural proteins result from the sequential action of the endoplasmic reticulum
(ER) resident enzymes signal peptidase (Hijikata et al., 1991) and signal peptide
peptidase (Weihofen et al., 2002). Processing of the non-structural protein region is
mediated by two virus-encoded proteases: The zinc-stimulated NS2-3 autoprotease
cleaves at the NS2/3 junction, and the NS3 serine protease is responsible for
liberating the remaining downstream non-structural proteins (Lindenbach and Rice,
2001). Efficient activity of the NS3 protease requires NS4A, a 54 amino acid-long
protein which acts as a cofactor for processing at the 3/4A, 4A/4B, 4B/5A, and
5A/5B sites (Bartenschlager et al., 1994; Failla et al., 1994; Tanji et al., 1995).
Stable partial cleavage products that include NS4B can also be detected during the
cleavage process (see Fig. 1 Bartenschlager et al., 1994). In other positive strand
RNA viruses, such partial cleavage products have independent activities, distinct
from those associated with the completely processed individual proteins (LaStarza

Fig. 1. Kinetics and possible partial cleavage products of HCV polyprotein processing. The first
cleavage event as indicated by the appearance of partial cleavage products is between NS3 and
NS4A. NS4A/B cleavage appears to be delayed as shown by the presence of the NS4A/B intermediate
and the slow production of NS4B. Two cleavage pathways seem to operate at the NS4B/5A site: A
rapid cleavage with production of the NS4A-B intermediate and a slow cleavage as indicated by the
presence of the relatively stable NS4A-4B-5A intermediate. Emphasis is placed on the generation
of NS4B. Other cleavage products are observed in cis and trans reactions. See Bartenschlager et al.,
1994, and Lin et al., 1994, for details.

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et al., 1994; Parsley et al., 1999). As described further below, there is evidence that
at least for one partially-processed precursor including NS4B, HCV may also make
use of such a strategy which increases the repertoire of functionally distinct protein-
encoded activities. Moreover, although many studies of NS4B examine the effects
of NS4B alone, NS4B may also function as part of multiprotein complexes with
NS4A and NS5A, with or without NS3 and NS5B (Lin et al., 1997; Neddermann
et al., 1999) (Fig. 1).

SUBCELLULAR LOCALIZATION AND TOPOLOGY

Initial studies of NS4B attempted to determine its subcellular localization as a
first step towards the understanding of its function. Indirect immunofluorescence
and green fluorescent protein (GFP) fusion experiments determined that NS4B is
cytoplasmically-localized in the perinuclear region where it adopts chickenwire-
like and speckled patterns typical of a membrane-associated protein (Kim et al.,
1999; Selby et al., 1993). Later Hugle et al. (Hugle et al., 2001) combined the use
of several methods, including specific antibodies and confocal analysis, to show
that NS4B was localized to the ER, where it colocalized with the other HCV
nonstructural (NS) proteins. This localization has been observed when the protein
was expressed either alone or in the context of HCV's other NS proteins, as well as
in cells harboring HCV replicons (El-Hage and Luo, 2003; Mottola et al., 2002).
Lundin et al. (Lundin et al., 2003) confirmed these localization results and reported
that the speckle or foci-like structures not only contained ER markers, but that
they tended to be both larger and more common the longer the cells are allowed
to express recombinant NS4B.

A recent study examined the localization of NS4B in live cells using a chimeric
NS4B-GFP fusion (Gretton et al., 2005). NS4B appeared to be distributed in
a thread-like pattern, consistent with ER localization, and at small foci similar
to those described by others (Gosert et al., 2003; Moradpour et al., 2004). The
authors termed these foci membrane-associated foci (MAFs). The mobility of
NS4B in ER membranes and MAFs was assessed using fluorescence recovery after
photobleaching (FRAP) experiments. In these experiments fluorescent molecules
in a defined area are irreversibly photobleached by a high-power laser. Subsequent
diffusion of non-bleached molecules into the bleached area leads to a recovery of
fluorescence. Fluorescence intensity in selected regions in live cells expressing the
NS4B-GFP protein was measured before and after photobleaching. A topologically
related GFP-tagged DNase X was used as a control. NS4B was determined to have
reduced mobility in MAFs compared with the ER membrane suggesting that NS4B
is likely to form different interactions on MAFs and the ER.

In vitro transcription–translation experiments performed in the presence

of microsomal membranes revealed that targeting to the ER membrane is

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cotranslational. Using classical membrane extraction and proteinase protection

assays it was shown that the majority of the protein is cytoplasmically oriented
but it was not possible to demonstrate the presence of transmembrane or lumenal
fragments (Hugle et al., 2001). This latter finding seemed somewhat puzzling
in light of other viral NS4B proteins having at least one transmembrane domain
(TMD) and that various computer predictions have estimated that HCV NS4B has
several TMDs. The inability to experimentally detect such TMDs could have been
for a variety of technical reasons and the lack of available antibodies to facilitate
the detection of such fragments.

Another approach to probing NS4B topology involved introducing canonical

glycosylation sites at various positions within NS4B. Here the rationale was that
the host cell enzymes responsible for glycosylation at such sites are exclusively
located within the ER lumen. Thus, those sites contained within NS4B segments
which are truly intralumenal would be expected to undergo glycosylation. Only two
of the predicted TMDs connected by a putative ER-lumenal loop could be supported
experimentally using this method (Lundin et al., 2003). It is possible, however, that
the extra amino acids introduced into NS4B in order to insert the target glycosylation
sites may have caused unanticipated deleterious changes to NS4B. Another study
found that all predicted TMDs could be deleted without impairing NS4B's ability
to associate with membranes (Elazar et al., 2004). Within the remaining segments
of NS4B, the authors detected a predicted amphipathic alpha helix domain at the
protein's N-terminus which was necessary for conferring membrane association
upon the mutant NS4B devoid of all TMDs (see more details below). Nevertheless,
the authors still favored a predicted NS4B topology containing TMDs, and suggested
that the membrane-associating function of the amphipathic helix was more likely to
mediate other membrane-associated functions of NS4B beyond simple anchorage to
membranes (Elazar et al., 2004). One such function may be to mediate a topologic
change of NS4B proposed to occur based upon an unexpected finding of the above-
mentioned glycosylation studies. Indeed, Lundin et al. (Lundin et al., 2003) observed
that a glycosylation site introduced into the NS4B N-terminal segment—predicted
to be cytoplasmically-oriented—was glycosylated in a manner to suggest that it
was translocated into the lumen in a fraction of the NS4B molecules. Such cases
of alternate topologies have been described in other viruses such as the hepatitis B
virus L envelope protein (Bruss et al., 1994) or the M protein from transmissible
gastroenteritis corona virus (Escors et al., 2001). The NS4B proteins of the yellow
fever and dengue viruses (Cahour et al., 1992; Lin et al., 1993) also have their N
termini located in the ER lumen due to an N-terminal signal peptide not found in
HCV NS4B. This potential shared topology might support the idea of a common
function for NS4B in Flaviviridae (Lundin et al., 2003).

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Although the above studies clearly show that NS4B behaves as a membrane-
associated protein, the precise nature of this association awaits further definition.
A variety of topologies with respect to the ER membrane have been proposed, with
NS4B predicted to have between 4-6 TMDs. The N and C termini are expected
to be (at least initially) located in the cytoplasm since they are generated by the
cytoplasmic NS3 protease (Hijikata et al., 1993; Wolk et al., 2000). Part of the
uncertainty stems from the fact that the computer algorithms used to predict
these topologies are derived from a databank of solved structures which contain
inadequate numbers of membrane proteins.

NS4B AND LIPID RAFTS

Lipid rafts are cholesterol- and sphingolipid-rich microdomains of cellular
membranes which are operationally-defined by their resistance to solubilization with
certain non-ionic detergents at 4°C (Cohen et al., 2004; Pike, 2004). Classically,
these detergent-resistant membrane microdomains and associated proteins
"float" to the low density upper fractions when subjected to density gradient
ultracentrifugation—hence the designation of rafts. Lipid rafts are known to play
important roles in diverse processes such as signal transduction and protein sorting
(Simons and Toomre, 2000; Slimane et al., 2003). Rafts are also exploited by an
increasingly recognized number of viruses as portals for viral entry or assembly
and release (Campbell et al., 2001; Cuadras and Greenberg, 2003; Manes et al.,
2003).

The first indication that HCV might also exploit lipid rafts was the demonstration
that both viral proteins and RNA could be detected in low density membrane
fractions resistant to solubilization with 1% NP-40 at 4°C (Shi et al., 2003). Although
their analysis was limited to NS5A and NS5B, these results suggested that the
membranes upon which HCV RNA replication occurs may be lipid rafts recruited
from intracellular membranes.

A more detailed analysis revealed that, when expressed together, NS proteins

3 through 5B could all be found in lipid raft fractions (Shi et al., 2003). When
expressed individually, however, only NS4B was completely associated with lipid
rafts (Gao et al., 2004). Therefore, NS4B, may be the key protein responsible for
binding to lipid rafts first and thereby enabling the recruitment or anchoring of
other NS proteins in order to form potential replication complexes.

This replication complex-harboring raft may be the same or different from that with
which the structural HCV core protein associates (Matto et al., 2004). Moreover,
although the raft targeted by NS4B shares biochemical features similar to those of
classical plasma membrane rafts, there appears to be some important differences.
For one, the steady-state cytoplasmic speckled staining pattern of NS4B is very

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different from the peripheral surface pattern typical of plasma membrane-based

rafts (Matto et al., 2004). Second, a significant amount of the raft-resident HCV NS
proteins and RNA are protected from digestion with exogenously added protease
and nuclease (Aizaki et al., 2004). This protection is lost upon treatment with
raft-solubilizing conditions. Taken together, these data suggest NS4B targets the
replication complex components to a specialized raft compartment which is not
directly contiguous with the host cell cytosol. It is tempting to speculate that this
compartment overlaps with the membranous web (to be described later) and that
the vesiculation and physical conformation of the latter helps provide some of
the observed protection from experimental nucleases. Similar protection may be
provided against host intracellular antiviral mechanisms (Barber, 2001).

THE IMMUNOLOGICAL EFFECTS OF NS4B

NS4B is recognized as a target by both the humoral and cellular arms of specific
immunity. Historically, NS4B sequences were contained in both the seroreactive
clone used to originally isolate the first fragment of the HCV genome (Choo et
al., 1989) as well as in the recombinant antigen used in the first generation of
anti-HCV commercial assays (Conry-Cantilena, 1997). Indeed some of the most
diagnostically relevant antigenic epitopes have been found to reside within NS4B
(Chang et al., 1999; Masalova et al., 2002; Rodriguez-Lopez et al., 1999) and this
helps explain why NS4B has since been successfully used as an antigenic target
in various commercial diagnostic tests for the detection of HCV antibodies in the
serum of patients with HCV infection. These highly antigenic properties might prove
beneficial for therapeutic purposes as well, such as using NS4B-derived peptides
to elicit an antiviral cytotoxic T lymphocytes (CTL) response.

To further characterize some of the mechanisms by which HCV induces an

inflammatory and immune response, Kato et al. (Kato et al., 2000) used HCV
viral protein– expression vectors cotransfected into mammalian cells with reporter
vectors having a luciferase gene driven by various cis-enhancer elements from 5
intracellular signaling pathways associated with cell proliferation, differentiation,
and apoptosis. Although core had the strongest effects, NS4B also significantly
activated the NF-κB–associated signal. Because the NF-κB pathway is known as
an inducer of inflammatory and immune responses, it was therefore suggested that
HCV core and NS4B proteins might modulate the production of various cytokines
and inflammatory responses in HCV-infected liver.

One of these cytokines appears to be interleukin-8 (IL-8). Indeed, serum IL-8 levels
have been shown to be elevated in patients infected with HCV (Polyak et al., 2001b)
and IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic
RNA replicon, compared with the control cells (Kadoya et al., 2005). Expression
of NS4B, (and to a lesser extent NS4A) alone were each found to significantly

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transactivate the IL-8 promoter, resulting in enhanced production of IL-8 protein.

The mechanism of IL-8 induction may be via NS4B's effect on NF-κB, as the IL-8
promoter contains a binding site for NF-κB. Because NS5A has also been implicated
in the induction of IL-8 (Polyak et al., 2001a), there would appear to be multiple
mechanisms whereby such induction can be induced by HCV.

As many cases of HCV are refractory to interferon (IFN) treatment, potential

mechanisms underlying HCV resistance to IFN have been the subject of intensive
investigation. One approach for studying such HCV resistance to IFN, centered
around developing IFN-resistant HCV replicons. For that, cells harboring HCV
replicons were subjected to a prolonged low-dose treatment with IFNs. Total RNA
derived from these IFN-treated replicon cells was then electroporated into naïve cells
and individual cell lines harboring HCV replicons with an IFN-resistant phenotype
were isolated. Here too, a possible role of NS4B was found. Indeed, sequencing
of the replicons contained in these cell lines revealed that they all shared a single
common amino acid substitution in NS4B (Q1737H) which might at least partially
explain their IFN-resistant phenotype (Namba et al., 2004). These results should
be interpreted with caution since cells cured from the replicon by cyclosporin A,
continued to show resistance to IFN suggesting that a host factor(s) rather than
replicon RNA(s) could have contributed to the IFN-resistant phenotype. Moreover,
direct demonstration that introduction of the Q1737H mutation into a wild type
replicon can confer IFN-resistance is still pending.

MODULATION OF NS5BS' RNA-DEPENDENT RNA

POLYMERASE ACTIVITY
It was recognized early on that HCV NS5B encodes the virus' RNA-dependent RNA
polymerase activity required for HCV replication. Many questions about how this
activity is controlled, however, remain unanswered.

To study the potential influence of NS3 and NS4B proteins on the priming activity of
NS5B, recombinant proteins were generated and introduced into an assay for NS5B's
RNA-dependent-RNA-polymerase (RdRp) activity on a template corresponding
to the minus strand 3'-untranslated region ((-)3'-UTR) (Piccininni et al., 2002).
Physical interactions between NS3 and NS5B as well as between NS3 and NS4B
were demonstrated. Both recombinant NS3 and NS4B proteins were also found to
modulate NS5B's RdRp activity, but in distinct ways: NS3, via its helicase function,
facilitated NS5B activity, whereas this effect was antagonized by the addition of
NS4B. These results provide additional evidence that NS4B can function as part of
a multi-protein replication complex. Although this data suggests that NS4B might
be a negative regulator of NS5B activity in vitro, this need not be the case in vivo
where additional regulatory factors may be operative.

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EFFECTS OF NS4B ON TRANSLATION

Since many viruses are known to interfere with host translational mechanisms,
possible inhibitory effects of HCV proteins on cellular protein synthesis were
analyzed using a transient expression system. The core protein, NS4A and NS4B,
but not NS3, NS5A or NS5B, inhibited the expression of cell cycle regulator protein
p21/Waf1/Cip1/Sdi1 (p21/Waf1) (Florese et al., 2002). There were no significant
differences in steady-state p21/Waf1 mRNA levels, as demonstrated by RT-PCR and
Northern blot analyses, suggesting the possibility of post-transcriptional inhibition.
That the inhibitory effect of NS4B may be at the level of translation was suggested
by in vitro translation assays which revealed inhibited synthesis of p21/Waf1
protein when co-translated with NS4B RNA. A similar inhibitory effect of NS4B
on the expression of RNaseL was detected although the magnitude appeared to be
somewhat smaller. It should be noted that a possible contribution of degradation
has not been ruled out.

Using a bicistronic reporter plasmid, the effects of HCV proteins on both cap-
mediated (host) and internal ribosome entry site (IRES)-mediated (virus) translation
were simultaneously monitored (Kato et al., 2002). In this system, the Renilla
luciferase is translated in a cap-dependent manner, while the firefly luciferase is
translated from the HCV IRES in a cap-independent manner. Both activities were
decreased with the expression of NS4A and/or NS4B proteins suggesting that NS4A
and NS4B proteins inhibited both cap-dependent translation and cap-independent
translation from HCV IRES. It was suggested that the latter might be a viral self-
regulation mechanism limiting the amount of viral protein. In contrast, using a
similar bicistronic reporter gene construct, IRES-mediated translation was found
to be specifically upregulated in HCV replicon cells (He et al., 2003). No such
enhancement was observed when the IRES from either poliovirus or EMCV were
substituted for the reporter construct's HCV IRES, suggesting specificity for HCV.
Transient expression of individual HCV non-structural proteins in combination
with the dual-luciferase reporter construct containing the HCV IRES showed that
NS5A and to a lesser extent NS4B could stimulate HCV IRES activity, although
the effect was less dramatic than in the context of the entire subgenomic replicon.
Reduced phosphorylation levels of both eIF2a and eIF4E were observed in the
replicon cells. In the absence of further mechanistic details whereby NS4B may
mediate its effects on translation, it is difficult to fully reconcile the above-detailed
differences observed by different investigators. Perhaps such discrepancies are due
to differences in duration and levels of expression of NS4B, or the presence of a
critical host cell factor.

"MEMBRANOUS WEB" FORMATION

A characteristic feature of plus-strand RNA viruses is their propensity to replicate
their genome in close association with host intracellular membranes. These

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membrane platforms can either be pre-existing membrane organelles or membrane

structures induced de novo by the virus (Chu and Westaway, 1992; Froshauer et
al., 1988; Lazarus and Barzilai, 1974; Rice, 1996). Egger et al. (Egger et al., 2002)
investigated by electron microscopy the capacity of HCV proteins to elicit such
intracellular membrane alterations by expressing HCV proteins individually or in
the context of the entire HCV polyprotein. Expression of the latter was associated
with the induction of a novel membrane structure designated "membranous web"
which appeared to consist of vesicles within a membranous matrix. Expression
of NS4B alone also induced the membranous web. The emergence of the latter
appeared to coincide with a reduction in the rough endoplasmic reticulum (RER),
and regions of continuity between the RER and membranous web were observed
(Egger et al., 2002). This suggested that the membranous web was derived from
the endoplasmic reticulum (ER). Similar structures have been described in livers of
HCV-infected chimpanzees (Pfeifer et al., 1980). Immuno-EM experiments revealed
that all examined HCV proteins could be found associated with the membranous
web, suggesting that these proteins might form a membrane-associated multi-
protein complex. Membranous web structures were also found in cells harboring
HCV replicons (Gosert et al., 2003). Importantly, viral plus-strand RNA could be
localized to these sites using a digoxigenin-labeled riboprobe followed by a gold
conjugated anti-digoxigenin antibody. Finally, nascent viral RNA synthesis detected
by metabolic labeling with BrU in the presence of actinomycin D (Gosert et al.,
2003) co-localized with immunofluorescently-detected NS5A. Similar apparent
co-localization of viral RNA and NS proteins has been described by others (Egger
et al., 2002; El-Hage and Luo, 2003; Shi et al., 2003). It was therefore postulated
that the membranous web represents the candidate site for HCV replication.

It remains to be clarified whether the membranous web, the MAFs described earlier
(Gretton et al., 2005), and the speckle-like structures detected by immunofluorescent
probes against HCV RNA and proteins in cells harboring subgenomic replicons
(Gosert et al., 2003) are all identical structures representing viral replication sites,
or different structures with different functions. It will also be important to confirm
the existence and character of membranous webs in cells which are capable of
permitting all aspects of the viral life cycle (Lindenbach et al., 2005; Wakita et al.,
2005; Zhong et al., 2005).

Nevertheless, NS4B's apparent key role in the establishment of the replication

(and possible early assembly) platform represented by the membranous web places
NS4B in a particularly prominent position in the viral life cycle. Moreover, because
the membranous web is not a normal feature of host cells, selective inhibition of
NS4B-induced membranous web formation could represent a specific antiviral
strategy with low inherent cytotoxicity.

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MODULATION OF ER FUNCTIONS
The ER is a major subcellular organelle with which the HCV life cycle is associated.
The HCV proteins are translated on, undergo initial processing by, and associate
with the ER. Moreover, as mentioned above, the membranous web is postulated
to be derived at least in part from the ER. It should therefore not be too surprising
that HCV in turn can affect a variety of ER functions.

In poliovirus, two different virally-encoded proteins slow the rate of ER-to-Golgi

traffic (Doedens and Kirkegaard, 1995) reducing the rate of ER protein secretion
and imparing the presentation of major histocompatibility complex class I (MHC-
I) antigens on the cell surface (Deitz et al., 2000). To determine whether HCV
proteins might induce similar effects, changes in anterograde traffic from the ER to
the Golgi apparatus were determined as a function of HCV NS protein expression
(Konan et al., 2003). For this, they monitored the glycosylation status of coexpressed
vesicular stomatitis virus G protein (VSV-G), a classical technique for secretory
pathway trafficking studies (Lodish et al., 1983). As VSV-G is transported from
the ER to the Golgi, the sensitivity of the covalently attached sugars to endo H
digestion is altered, thus providing a convenient marker of anterograde trafficking.
Of all the NS proteins evaluated, including putative partially-processed precursor
proteins, only a fused NS4A/B affected the rate of ER-to-Golgi traffic, reducing it by
approximately three fold. Interestingly, no effect was seen with the fully-processed
products NS4A or NS4B alone, or in combination. NS4A/B expression inhibited
the secretion of other cargo proteins as well. In cells harboring full length HCV
replicons, MHC-I appearance on the cell surface was attenuated by three- to five
fold compared to control cells. Both NS4A/B and NS4B caused the accumulation
of clustered, aggregated membranes in 293T cells and were found localized to
these membranes. Only NS4A/B caused the formation of swollen vesicles, but the
protein did not localize to these structures. These swollen vesicles were suggested
to be ER-derived membranes swollen with cargo due to the blockage in ER-to-
Golgi traffic. It was postulated that such blockage –with the associated reduction of
cytokine secretion and transport of membrane proteins such as MHC-I to the cell
surface--could affect the host immune response to HCV infection.

HCV has also been implicated in the induction of ER stress (for review see Tardif
et al., 2005) and this may contribute as well to the decrease in MHC-I expression
found in cells harboring HCV replicons (Tardif and Siddiqui, 2003). NS4B may
play both a role in the induction of ER stress and its regulation. One mechanism
may reside with the ATF6 (activating transcription factor 6) activation associated
with HCV replication (Tardif et al., 2002). ATF6 is a transcription factor activated
to alleviate ER stress when protein folding is disrupted. Using a yeast two-hybrid
assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP),
also called ATF6β, was identified to interact with NS4B (Tong et al., 2002). The

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N-terminal half of NS4B and a central portion of CREB-RP/ATF6β containing

the basic leucine zipper (bZIP) domain were involved in this interaction. ATF6α,
which shares high sequence similarity with CREB-RP/ATF6β, was also shown to
interact with NS4B in yeast although the interaction was weaker than that between
NS4B and CREB-RP/ATF6α. Interestingly, ATF6β suppresses transcription of ER
stress-inducible genes while ATF6α enhances it (Thuerauf et al., 2004). This might
suggest that NS4B can, like NS5A and E2 (Gale et al., 1997; Pavio et al., 2003;
Taylor et al., 1999), inhibit specific downstream pathways of ER stress induction.
At present, however, interactions of NS4B with ATF6 in vivo and the functional
consequences remain to be determined. Finally, it is possible that some of the above
effects are consequences of NS4B's interactions with membranes per se, including
the diversion of ER components into the creation of the membranous web.

MALIGNANT TRANSFORMATION
The leading cause of hepatocellular carcinoma (HCC) in the US is hepatitis C virus.
Typically, this severe complication of HCV occurs many years after infection, in the
setting of cirrhosis. Because the latter is an independent risk factor for HCC, HCV-
associated HCC could either be simply an indirect consequence of HCV-induced
cirrhosis. Alternatively, the HCC could be the direct result of specific viral factors,
presumably in the context of a "multi-hit" scenario where the time course for full
accumulation of these hits parallels the development of cirrhosis. In the context
of the latter possibility, several HCV proteins including the core protein and non
structural proteins NS3 and NS5A have been reported to transform various cell lines,
and in the case of core cause tumors when expressed in transgenic mice (Moriya et
al., 1998). The cell transformations occur either alone or in cooperation with other
known oncogenes (Ghosh et al., 1999; Ray et al., 1996; Ray et al., 2000; Sakamuro
et al., 1995). The involvement of HCV's NS protein NS4B in tumor formation was
also investigated (Park et al., 2000). NIH3T3 cells co-transfected with NS4B and
the Ha-Ras gene showed loss of contact inhibition, morphological alterations, and
anchorage-independent growth--all characteristics of a transformed phenotype.
Similar experiments using c-src, c-fos , c-myc substituted for the Ha-ras, failed
to show any tumorigenic phenotypes, suggesting a specificity for enhancement of
Ras-mediated pathways. Since many viral proteins are involved in Ras-mediated
transcriptional regulation and growth control through AP1 activation, the effect of
NS4B on luciferease activity controlled by the AP1 promoter was examined (Park
et al., 2000). The luciferase gene was cloned under the control of the AP1 promoter
and transfected into NIH3T3 cells stably co-transfected with NS4B and Ha-ras.
Luciferase activity in these cells was increased by six fold in comparison with
cells stably transfected with Ha-ras alone. AP1-Luc transfection into stable NS4B
transfectants did not increase AP1-Luc activity. This suggests that the apparent
synergy between NS4B and Ha-ras might be mediated via AP1 activation. Because
of the limitations associated with interpreting experiments involving overexpression

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and in vitro transformation correlates, the relevance of the above (albeit provocative)
observations to clinical HCV-associated HCC remains to be determined

NS4B FEATURES THAT MAY UNDERLIE THE MECHANISMS

OF THE ABOVE FUNCTIONS

THE NS4B AMPHIPATHIC HELIX

Similar to NS5A (Elazar et al., 2003), NS4B has a predicted N-terminal amphipathic
helix (see Fig. 2) which suggested another mechanism of membrane association
in addition to NS4B's TMDs (Elazar et al., 2004). This amphipathic helix (AH)
was found to be conserved across all HCV isolates, suggesting it plays a critical
role in productive natural infections. Introduction of mutations designed to
disrupt the hydrophobic face of the AH abolished its ability to mediate membrane
association.

This disruption abolished HCV RNA replication, whereas mutations designed to

only partially disrupt the amphipathic nature of the AH resulted in an intermediate
level of replication (Elazar et al., 2004). These results genetically validate the NS4B
AH as a potential antiviral target, although the mechanistic details underlying the
NS4B AH's critical role in replication await further definition. One possibility
may be to help mediate the establishment of the HCV replication complex.

Fig. 2. The N-terminus of NS4B harbors a predicted amphipathic

helix. The amino-terminal segment of NS4B is predicted to
adopt an alpha helical secondary structure, depicted here in a
helix net diagram wherein the cylindrical alpha-helical segment
is "sliced" longitudinally along one face and "flattened" into the
plane of the page. Amino acids in the N- to C-terminal direction
is shown beginning at proline 5. Hydrophobic amino acids are
shaded in grey. Note the continuous stretch of such amino acids
along one side of the helix, defining its amphipathic nature.

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 The HCV Non-structural Protein 4B

When NS4B is expressed from a subgenomic replicon with a mutated NS4B AH,
localization of NS4B is aberrant and the cytoplasmic speckle-like pattern typical
of wild type replicon cells is lost (Elazar et al., 2004). The mutant NS4B retains a
reticular staining pattern suggestive of ER localization, but it is unable to be further
sublocalized into the characteristic speckles. Moreover, not only is normal NS4B
localization abrogated, but the disrupted NS4B AH prevents other members of the
HCV replication complex form coalescing into the speckled pattern associated
with replication-competent replicons. Thus the NS4B AH may be responsible for
mediating the association of NS4B and replication complex components with lipid
rafts. The AH is also hypothesized to play a role in membranous web formation.
Interestingly, a second AH has also been identified within NS4B (Glenn and Elazar,
unpublished data), which may also play an important role in the viral life cycle.

NS4B HAS A NUCLEOTIDE BINDING MOTIF

Inspection of the NS4B primary sequence revealed the presence of a candidate
nucleotide binding motif (NBM) beginning in the middle of the protein. Such NBMs
are characterized by conserved of sets amino acids present in proteins known to
bind nucleotides. The most conserved elements of NBMs are the so-called A motif
(GxxxxGK) and B motif (DxxA) which are separated by a variable number of
amino acids, depending on the particular protein (Gorbalenya and Koonin, 1989).
Additional motifs common in a large number of GTP-binding proteins, such as the
G-protein superfamily, can be identified. Among these are the G and PM2 motifs
consisting of single amino acids (F and T, respectively) located between the A and
B motifs (Fig. 3).

The crystal structures of several G-proteins has revealed that the G and PM2
elements interact with the nucleotide base (guanine in the case of G-proteins) and
the chelated Mg++ ion, respectively (Stenmark and Olkkonen, 2001).

NS4B was found to specifically bind GTP (Einav et al., 2004). Similar to many
other nucleotide-binding proteins, NS4B was also able to hydrolyze nucleotide,
indicating it is a GTPase. Mutations disrupting the A motif element of the NBM
impaired GTP binding and hydrolysis. These same mutations dramatically inhibited
HCV RNA replication, and the effects on GTPase activity paralleled the effect on
replication (Einav et al., 2004). Further mutagenesis experiments disrupting the
B and the G motifs showed similar effects on viral replication (Moon and Glenn
unpublished results). None of these mutations had any apparent effect on NS4B
protein levels or its targeting to the ER. Together these results suggest that the
nucleotide binding motif within NS4B is essential for mediating NS4B's role in HCV
replication in vitro. The requirement of a nucleotide binding motif for productive
viral infection in vivo is further suggested by the conservation of this motif across
natural HCV isolates of all genotypes. Although it is clear that this NBM mediates

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Fig. 3. Elements of NS4B's nucleotide binding motif (NBM). The NS4B protein is depicted
schematically with its 4 predicted TMDs in relation to the ER membrane. The relative positions and
amino acid composition of the A motif, B motif, G and PM2 motifs are indicated (black)—together
these elements constitute the NS4B NBM. Also noted is the position of the amphipathic helix (white).
Numbers correspond to amino acid positions. See text for details.

critical functions in the viral life cycle the exact details of its function await
further definition. One possibility can be that the NS4B NBM mediates binding
of nucleotides not only as single molecules but also as part of a polynucleotide
structure such as RNA. By simultaneously binding cellular membranes and RNA,
NS4B might contribute to the structural integrity of the replication complex by
helping to anchor it to membranes.

The ability to bind and hydrolyze GTP has evolved to serve diverse regulatory roles
in biology, in part because it represents an efficient and regulateable molecular
switch. As such, the NS4B NBM affords a wide variety of potential regulatory
mechanisms and it can be readily envisaged to mediate many of the effects ascribed
to NS4B in this chapter. Because the amino acids upstream and downstream of the
NBM are highly conserved across HCV isolates, yet very different from known
host cell G-proteins, there is also the potential for selective inhibition of the NS4B
NBM.

FUTURE DIRECTIONS
As reviewed in the preceding sections, mounting evidence indicates the importance
of NS4B to various viral activities. NS4B also appears to be connected with various
viral effects on the host cell. It is quite clear that to mediate all these effects NS4B

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likely has a variety of cellular and/or viral protein partner(s). Uncovering their
identity may further clarify some of NS4B's functions—many of which still have
unproven mechanisms. Important information might be gained from investigating
common features in the NS4B proteins of different viruses from the Flaviviridae
family. For example, the related bovine viral diarrhea virus isolates divide into
cytopathic and noncytopathic biotypes. In all noncytopathic biotypes that arouse
from cytophatic variants an Y2441C substitution in NS4B was found. This might
implicate the involvement of NS4B in viral cytopathogenicity (Qu et al., 2001).

Although information about NS4B is continuing to accumulate, several key points

relating to its currently ascribed functions remain unclear. For example, only 2 of the
four to six predicted TMDs in NS4B have experimental validation. Understanding
the exact topology of NS4B could assist in further revealing some of its functions
and in the design of specific inhibitors. Another issue awaiting further clarification is
the exact intracellular localization of NS4B and its relationship to viral replication.
The NS4B-induced membranous webs, MAFs, and the characteristic NS4B speckles
may or may not be the same structures. Moreover, which of these represents the
authentic sites of viral replication remains to be clarified. NS4B's inhibitory effects
on the host translation machinery seem somewhat clear but its exact effect on viral
IRES-mediated translation remains uncertain. Improving the understanding of
these issues might provide the requisite tools to specifically control viral protein
translation. Similarly, the critical role of NS4B's NBM in the viral life cycle has
been demonstrated but the exact details of the function(s) mediated by the GTPase
activity remain to be fully described. Although there are undoubtedly yet to be
discovered features of NS4B, its already identified properties clearly make it a
valuable probe of host cell biology.

Both the NS4B AH and NBM provide potential mechanisms to mediate many of
the proposed functions for NS4B. The ability to pharmacologically inhibit these
domains thus represents another exciting avenue for future research. With respect
to the AH, similar strategies as those shown to be effective against the NS5A AH
(Elazar et al., 2003) can be readily adapted to the NS4B AH target. The NBM may
offer even more readily adaptable antiviral strategies.

Further characterization of the possible role of NS4B in malignant transformation

may advance the understanding of HCV-associated carcinogenesis mechanisms
and may lead to novel therapeutic strategies. Alternatively, effective pharmacologic
eradication of HCV could by itself make the leading cause of hepatocellular
carcinoma in the US theoretically preventable. By analogy with other infections,
such as tuberculosis or HIV, this type of pharmacotherapy is most likely to consist
of a cocktail which includes multiple agents, each designed against an independent
virus-specific target. Exploitation of current and yet to be identified targets within

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NS4B could increase the repertoire of agents available for inclusion in such
therapeutic cocktails of the future.

FUTURE DIRECTIONS
This work was supported by ROIDK066793 and Burroughs Wellcome Career
Award (to JSG).

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Chapter 9

HCV NS5A: A Multifunctional Regulator of

Cellular Pathways and Virus Replication
Yupeng He, Kirk A. Staschke and Seng-Lai Tan

ABSTRACT
The hepatitis C virus (HCV) non-structural 5A (NS5A) protein has generated wide
interest in HCV research because of its ability to modulate the host cell interferon
(IFN) response. The protein is phosphorylated on multiple sites by host cell kinases
and interacts with host cell membranes. While no known enzymatic function has
been ascribed to NS5A, it is an essential component of the HCV replicase and
exerts a wide range of effects on cellular pathways and processes, including innate
immunity and host cell growth and proliferation. In this chapter, we review the
many studies describing the interaction of NS5A with viral and host cell proteins, its
ability to modulate multiple cellular pathways, and its recently described structural
attributes, subcellular localization, and function during HCV replication.

INTRODUCTION
Translation of the HCV genome results in the production of a large polyprotein,
from which NS5A is processed by the NS3 protease (Reed and Rice, 2000). As a
nonstructural (NS) protein with no apparent enzymatic activity, NS5A functions
through interaction with other viral and cellular proteins. Its primary amino acid
(a.a.) sequence predicts a proline-rich, predominantly hydrophilic protein with no
obvious trans-membrane helices. NS5A exists as multiple phospho-isoforms and
is predominantly localized in the cytoplasmic/perinuclear compartments of the
cell, including the ER and the Golgi apparatus. This pattern of NS5A localization
is consistent with the notion that NS5A interacts with multiple host cell and viral
proteins. There is strong evidence that NS5A is also localized in certain modified
cytoplasmic membrane structures during HCV replication, where it plays a
functionally significant role as part of the HCV replication complex or replicase.
NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral
replication, host-cell interactions, and viral pathogenesis.

STRUCTURAL FEATURES AND SUBCELLULAR

LOCALIZATION OF NS5A
Early studies utilizing cells in which NS5A had been overexpressed or liver
biopsy samples from chronic HCV patients, showed that NS5A is localized in the

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cytoplasm and the perinuclear membrane fraction, consistent with localization to

the ER/Golgi (Ide et al., 1996; Polyak et al., 1999; Tanji et al., 1995a). In addition,
when expressed either alone or in the context of additional HCV NS proteins (an
NS3-5B polyprotein) in human hepatoma cells, NS5A was also found to co-localize
with the HCV core protein on the surface of globular structures containing lipid
droplets (Shi et al., 2002). Interestingly, previous studies had noted that NS5A
binds to the core protein on membrane structures (Goh et al., 2001). Also, NS5A
was found to bind to Apolipoprotein A1 (ApoA1), a protein component of high-
density lipoprotein (HDL) particles and co-localized with ApoA1 in the Golgi (Shi
et al., 2002). In other studies, NS5A was found to bind to a snare-like protein called
hVAP-33 (Tu et al., 1999). In HCV replicon cells, NS5A bound to hVAP-33 and
localized to detergent-resistant lipid rafts (Gao et al., 2004). In a study utilizing
an HCV replicon in which NS5A was fused to green fluorescent protein (GFP),
the NS5A-GFP fusion protein was associated with brightly fluorescent dot-like
structures in the cytoplasm (Moradpour et al., 2004). Analysis of these structures by
electron microscopy led to their description as "membranous webs". It was suggested
that these might represent sites of bound replication complexes or sites of virus
assembly since HCV NS proteins and nascent viral RNA all co-localized to these
structures (Moradpour et al., 2004). Given its interaction with host cell proteins and
membranes, as well as the HCV core protein (Goh et al., 2001), it is possible that
NS5A may also regulate HCV virus assembly directly through its interaction with
the viral capsid protein. So it seems that after its expression and processing in the
ER, the NS5A protein localizes into specialized cytoplasmic membrane structures,
as a part of putative HCV replication and/or assembly complexes. These specialized
membrane structures may be derived from or related to either ER or Golgi, and
the association of NS5A with these structures may require interaction with host-
derived, membrane-associated proteins or other viral proteins. The importance of
cellular membranes is underscored by the recent finding that inhibitors of protein
geranylgeranylation (Ye et al., 2003) and fatty acid biosynthesis (Kapadia, 2005)
block HCV replication in replicon cells. Recently, FBL-2, a geranylgeranylated
cellular protein was shown to bind to NS5A and found to be critical for HCV
replication (Wang et al., 2005). Whether or not additional cellular proteins play a
role in these processes is not known.

The membrane association of NS5A protein occurs post-translationally and NS5A

has similar properties to that of an integral membrane protein (Brass et al., 2002). A
membrane-anchoring region was mapped to the N-terminal 30 a.a. of NS5A which
form a highly conserved amphipathic α-helix (Brass et al., 2002). It was suggested
that membrane anchorage is mediated by the hydrophobic side of the amphipathic
helix, resulting in an orientation parallel to the lipid bilayer, while positioning the
helix in the cytoplasmic leaflet of the ER membrane. A follow-up study demonstrated
that the N-terminal amphipathic helix is not only necessary and sufficient for

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membrane localization, but also important for HCV replication since mutations
disrupting helix formation impaired HCV replication (Elazar et al., 2003). A three-
dimensional structure of this region was solved by NMR spectroscopy (Penin et
al., 2004). The structure revealed an α-helix extending from a.a. 5 to 25. The helix
contains a hydrophobic side embedded in detergent micelles, and a solvent-exposed,
polar, charged side. Confirmatory studies showed that the NS5A membrane anchor
region forms an in-plate, amphipathic α-helix, embedded in the cytosolic leaflet of
the membrane bilayer. It was also suggested that this region is not only involved in
membrane localization, but also required for additional functions, as mutations of
conserved residues on the cytosolic face impaired HCV replicon RNA replication
without affecting membrane association (Penin et al., 2004). In addition to its role
in localizing NS5A protein to appropriate membrane compartments, it is likely that
the membrane anchor region provides a platform for protein-protein interactions
involved in the HCV replication process. It is also possible that specific cytosolic
residues of the helix may contribute to protein-protein interactions with additional
viral and cellular components.

Until recently, structural information on the NS5A protein had been limited, largely
due to the difficulty in purifying the full-length protein. Early studies on NS5A
structure were limited to individual structural motifs and their functions. With the
recent characterization of its domain organization and resolution of a structure
of the N-terminal region (Tellinghuisen et al., 2004; Tellinghuisen et al., 2005),
we can begin to gain an appreciation for the multi-dimensional structure of the
NS5A protein. A recent study using bioinformatics-assisted modeling suggested a
three-domain organization (Tellinghuisen et al., 2004) with domain I (a.a. 1-213)
located in the N-terminal region, and Domain II (a.a. 250-342) and Domain III
(a.a. 356-447) in the C-terminal region (Fig. 1). This organization was confirmed
by limited proteolysis experiments. Interestingly, an unconventional zinc-binding
motif was predicted to exist in the N-terminal domain, indicating that NS5A is a
zinc metalloprotein (Tellinghuisen et al., 2004). The predicted zinc-binding motif
involves four cysteine residues (C39, C57, C59, and C80; Fig. 1), and includes a
structural motif (CX17CXCX20C) that is well conserved among Hepaciviruses
and Pestiviruses. In this same study, the zinc content of purified NS5A protein or
the N-terminal domain alone was determined and it was found that each protein
molecule coordinates one zinc atom. This motif appeared critical for the structural
stability and function of the NS5A protein, since mutation of any single cysteine
residue in the motif disrupted the ability of NS5A to coordinate zinc and eliminated
HCV replicon RNA replication (Tellinghuisen et al., 2004). A more recent study
reported the crystal structure of NS5A Domain I (a.a. 36-198) at 2.5-A resolution
(Tellinghuisen et al., 2005). The structure revealed the presence of a novel fold,
a zinc-coordination motif, and a C-terminal disulfide bond. Mutational analysis
suggested that the disulfide bond is not required for the HCV replicase functions of

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Fig. 1. Schematic diagram of the NS5A protein. Several prominent features of the NS5A protein and
described in detail in this review are shown. The three domain structure of NS5A (Tellinghuisen et
al., 2005) is depicted. The N-terminal amphipathic α-helix (Elazar et al., 2003; Penin et al., 2004),
the IFN sensitivity determining region (ISDR) (Tan and Katze, 2001), the class I and class II proline-
rich (Tan et al., 1999), and NLS sequences (Ide et al., 1996) are also shown. In addition, basal and
hyperphosphorylation sites as well as the binding sites for several interacting proteins (see Table 1)
are noted.

NS5A (Tellinghuisen et al., 2004). These studies have provided a nice starting point
for understanding the structural organization of NS5A, the elucidation of structural
assembly points of NS5A as it pertains to its role as an HCV replicase subunit, and
NS5A's ability to interact with multiple host cell proteins and molecules.

While most studies have focused on the membrane associated forms of NS5A,
an early study identified a putative nuclear localization signal (NLS) sequence
(PPRKKRTVV; a.a. 354-362) within the C-terminal half of NS5A (Ide et al.,
1996) (Fig. 1). This sequence appeared to function as an NLS since it was able
to target a heterologous protein (β-Galactosidase of E. coli) into the nucleus.
The presence of an NLS suggests a possible nuclear localization and function of
NS5A in addition to its membrane bound isoforms. One study suggested that the
localization of NS5A to membranes is at least partially determined by its most N-

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terminal region (Satoh et al., 2000). It was found that NS5A mutants lacking this
region were localized in the nucleus. Conversely, the N-terminal 27 a.a. from NS5A
were capable of retaining a nuclear protein in the cytoplasm. In addition, a cleaved
form of NS5A protein missing the N-terminal region (a.a. 155-389) also localized
to the nucleus. The N-terminal sequence was able to block the function of the NLS
in the C-terminal region and prevented NS5A protein from being transported into
the nucleus (Song et al., 2000). This putative "NLS-masking-sequence" in the
N-terminus, which appears to overlap with the amphipathic α-helical region, did
not function as a nuclear export signal. So it seems that the this region can also
regulate the function of the NLS and thus the nuclear localization of NS5A protein,
presumably by preferentially targeting NS5A protein into cytoplasmic membrane
structures. It is likely that the localization of NS5A protein in different subcellular
compartments is determined and regulated by different structural features and/or
different forms of the protein, and the differential localization of NS5A in different
compartments may contribute to its different biological functions. In particular,
the cytoplasmic vs. nuclear localization and function of NS5A could be carefully
counter-regulated and balanced through its different structural motifs regulating
subcellular localization of the protein during the viral life cycle. Along these lines,
the C-terminal half of NS5A contains a positively charged region enriched with
acidic and proline residues, a structural feature resembling those of eukaryotic
transcriptional activators (Chung et al., 1997; Ide et al., 1996). Following deletion
of the N-terminal membrane anchoring domain, the C-terminal half of NS5A
functioned as a potent transcriptional activator when fused to the DNA-binding
domain of yeast GAL4 protein, in both yeast and human hepatoma cells (Chung et
al., 1997; Kato et al., 1997; Tanimoto et al., 1997). Furthermore, a region between
a.a. 130-352 was found to be critical for optimal transcriptional activation (Tanimoto
et al., 1997). These studies suggest that truncated forms of NS5A may localize to
the nucleus via the cryptic NLS only after removal of the N-terminal membrane-
anchoring region and regulate cellular gene transcription. The mechanism of NS5A
nuclear localization may involve proteolytic processing of NS5A. Indeed, this was
observed and a cleaved form of the protein was able to localize to the nucleus and
caused transcriptional activation when the alpha subunit of PKA was co-expressed
(Satoh et al., 2000; Song et al., 2000). The NS5A cleavage in mammalian cells was
enhanced by apoptotic stimuli and was inhibited by the caspase inhibitor Z-VAD-
FMK, suggesting that a caspase-like protease(s) contributes to the cleavage of
NS5A (Satoh et al., 2000). A later study showed that NS5A protein was also cleaved
following induction of apoptosis by the HCV core protein and that the proteolytic
processing of NS5A could be inhibited by Z-VAD-FMK (Goh et al., 2001). These
studies indicated that NS5A protein cleavage is likely mediated by caspase(s) and/or
related protease(s) and may be linked to the induction of apoptosis. In support of
this, a recent study found that NS5A was processed into multiple forms in different
mammalian cell types (Vero, HepG2, Huh-7, and WRL68), and suggested that both

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caspase-like proteases and calcium-dependent calpain proteases were involved in

NS5A processing (Kalamvoki and Mavromara, 2004). However, this study also
showed that both the cleaved and full-length forms of NS5A exhibited a cytoplasmic/
perinuclear localization. Although these results suggest that additional proteolytic
processing of NS5A may occur, the biological function the cleaved forms in the
context of HCV biology remain uncertain at the moment.

NS5A PHOSPHORYLATION: A FUNCTIONAL ROLE OR RED

HERRING?
Studies on NS5A expressed in tissue culture revealed predominantly two
forms of NS5A protein with differing apparent molecular weights of 56 and 58
kDa. Basal phosphorylation results in expression of the 56 kDa isoform while
hyperphosphorylation results in the 58 kDa form(Kaneko et al., 1994; Tanji et al.,
1995b). In addition, there is evidence that p58 is converted from p56 and requires
polyprotein processing (Neddermann et al., 1999). Phosphorylation of NS5A protein
occurs predominantly on serine residues, with a minor fraction on threonine residues
(Kaneko et al., 1994; Reed et al., 1997; Tanji et al., 1995b). A number of serine
residues (2194, 2197, 2201, and/or 2204) in the central region of NS5A were found
to be important for hyper-phosphorylation, and two other regions (a.a. 2200-2250
and the C-terminal region) appeared important for basal-phosphorylation (Tanji
et al., 1995b) (Fig. 1). In addition, a major phosphorylation site was identified as
serine 2321, which is located within the C-terminal Class II proline-motifs and
likely represents a basal phosphorylation site (Reed and Rice, 1999). In another
study, Katze and colleagues identified the major phosphorylated residue on an
NS5A phosphopeptide (a.a. 2193-2212) as serine 2194, which is well conserved
among HCV genotypes and presumably is a site for hyper-phosphorylation (Katze
et al., 2000). Additional phosphorylation sites remain to be mapped. In addition,
phosphorylation of NS5A on tyrosine has not been reported. Interestingly, NS5A
proteins from other viruses closely related to HCV, such as BVDV and YFV, were
also phosphorylated in various in vitro and in vivo systems, and it appeared that
phosphorylation occurred via serine/threonine kinase(s) (Reed et al., 1998). These
results indicate that NS5A phosphorylation is a well-conserved feature, and either
the phosphorylation of NS5A itself or NS5A interaction with its cellular kinases
plays an important role in the Flavivirus life cycle. Whether these NS5A proteins
from the different virus species are phosphorylated by the same or related kinase(s)
is completely unknown. However, one study reported that NS5A protein from
HCV genotype-2a was not hyperphosphorylated in contrast to that of NS5A from
genotype-1 (Hirota et al., 1999). This raises the question as to whether the same
phosphorylation pattern is prevalent throughout all HCV genotypes/isolates, and
whether different phosphorylated forms of the NS5A protein play different roles
in viral pathogenesis or in the HCV viral life cycle. It is also possible that the
different NS5A phosphorylation patterns in vitro were caused by differences in

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the experimental systems employed. Unfortunately, due to technical limitations,

the phosphorylation of NS5A in the liver of chronic HCV patients is not easily
addressed.

Numerous studies have attempted to identify the cellular kinase(s) responsible

for NS5A phosphorylation. NS5A protein was found to stably associate with an
unknown protein kinase(s) from mammalian cells, and this kinase was able to
phosphorylate native NS5A protein on serine residues in vitro (Ide et al., 1997).
This same study also showed that the catalytic subunit of cAMP-dependent protein
kinase A (PKA) was capable of phosphorylating NS5A in vitro. Interestingly, as
previously noted, co-expression of the alpha subunit of PKA seemed to affect the
transcriptional activity of a cleaved form of NS5A (Satoh et al., 2000). However,
there is no evidence that PKA is an NS5A kinase in mammalian cells. By testing
the effect of various kinase inhibitors on NS5A phosphorylation in vitro and
examining the context of known phosphorylation sites, other studies suggested
that the NS5A kinase(s) belongs to the CMGC group of serine-threonine kinases
and is likely a proline-directed kinase (Katze et al., 2000; Reed and Rice, 1999;
Reed et al., 1997). Indeed, casein kinase II (CK II), a member of the CMGC kinase
family, was found to phosphorylate NS5A protein in vitro, and it showed the same
molecular size and properties as an unknown kinase that stably associates with
NS5A in mammalian cells through the N-terminal region of NS5A (Kim et al.,
1999). Thus, CK II stands as a candidate kinase for NS5A phosphorylation, but
direct evidence for its role in vivo remains elusive. A more systematic approach
was employed by Coito and colleagues, who performed a global screening of all
yeast kinases capable of phosphorylating NS5A in vitro, and then attempted to
predict and identify homologous mammalian kinases that were also capable of
phosphorylating NS5A through both bioinformatic and biochemical methods (Coito
et al., 2004). By comparing in vivo and in vitro NS5A phosphopeptide profiles,
their results suggested that several mammalian kinases (AKT, p70S6K, MEK1,
and MKK6) might be responsible for NS5A phosphorylation in vivo. In particular,
the functional relevance of p70S6K or related kinases was further supported by
the fact that rapamycin was able to reduce the phosphorylation of specific NS5A
phosphopeptides in vivo. Given the complexity of NS5A phosphorylation, it is likely
that multiple kinases are involved and that phosphorylation occurs in a regulated
and coordinated manner.

Several lines of evidence suggest that the pattern NS5A phosphorylation is

dependent on additional HCV NS proteins. One group reported that the level of
the hyperphosphorylated form of NS5A (p58), was enhanced by the presence of
NS4A. Additionally, the association of NS5A with NS4A through a.a. 2135-2139
of NS5A was important for NS4A-dependent phosphorylation (Asabe et al., 1997;
Kaneko et al., 1994). A later study suggested that the appearance of p58 required

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NS2 in cis and the autoproteolytic activity of the NS2-3 protease. The loss of
p58 by disruption of NS2-3 autoproteolysis was rescued by expressing an NS2-
3 in trans (Liu et al., 1999). However, other studies published at the same time
showed that the presence of NS3-4A-4B in cis was necessary and sufficient for the
hyperphosphorylation of NS5A (Koch and Bartenschlager, 1999; Neddermann et al.,
1999) and the presence of NS3-4A protease activity in cis was absolutely required
for p58 production (Neddermann et al., 1999). Interestingly, it was also found that
single a.a. mutations with NS3, as well as mutations within NS4A and NS4B that
do not disrupt polyprotein processing, also affected NS5A hyperphosphorylation
(Koch and Bartenschlager, 1999). In summary, the exact requirement for other HCV
NS proteins and their roles in NS5A phosphorylation is not completely understood,
but it seems likely that NS5A phosphorylation is regulated in the context of other
NS proteins, and requires both polyprotein processing and interactions among the
NS proteins within a multi-subunit protein complex. Despite these observations,
only recently has the role of NS5A phosphorylation been described in the context
of HCV replication (Evans et al., 2004). These latter studies suggest that the
differential phosphorylation of NS5A regulates its function during HCV replication,
presumably by affecting its interaction and formation of protein complexes with
other proteins.

EMERGING ROLE OF NS5A IN HCV REPLICATION

Studies utilizing subgenomic HCV replicons in cell culture systems suggest
that NS5A plays an important role in the establishment of high-level HCV RNA
replication (see Chapter 11). Several adaptive mutations that confer higher
replication efficiency to HCV replicons are clustered in the NS5A region, and some
of these adaptive mutation sites either overlap with putative NS5A phosphorylation
sites or have been shown to affect NS5A hyperphosphorylation. This re-opened
the question as to whether NS5A phosphorylation plays a role in HCV replication.
Interestingly, when expressed alone in mammalian cells in culture, NS5A has an
apparent half-life of four to six hours (Polyak et al., 1999). In replicon cells, the
hyperphosphorylated (p58) form of NS5A is much less stable than the basally
phosphorylated (p56) form of the protein (Pietschmann et al., 2001), suggesting
possible differences in function. Indeed, one study found an inverse relationship
between NS5A phosphorylation level and its interaction with hVAP-33, which in
turn, is required for HCV replication in the replicon system (Evans et al., 2004). In
addition, some of the previously identified adaptive mutations suppressed NS5A
hyperphosphorylation and increased NS5A binding to hVAP-33. It is noteworthy
that the region of NS5A that interacts with hVAP-33 encompasses the putative
NS5A hyperphosphorylation sites (Fig. 1). NS5A also binds directly to the NS5B
viral polymerase both in vitro and in vivo (Shirota et al., 2002). This interaction was
suggested to modulate the enzymatic activity of NS5B (Shirota et al., 2002) and in
replicon cells shown to be critical for HCV replication (Shimakami et al., 2004). A

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model has been proposed in which the phosphorylation status of NS5A serves as a
molecular switch in the regulatory process of HCV RNA replication by affecting the
association between NS5A and other components of the viral replication complex
(Evans et al., 2004). This model also implies that host cell kinases regulate the HCV
replication process through differential NS5A phosphorylation. In line with this
working model, another study identified three undisclosed kinase inhibitors that
blocked NS5A hyperphosphorylation in cell culture, and showed that treatment with
any of these compounds stimulated replication of a wild-type replicon construct
that has no adaptive mutations and replicates poorly otherwise (Neddermann et
al., 2004). This is an exciting finding since this approach might allow efficient
replication of many HCV strains that otherwise replicate very poorly in cell culture.
This method may also open a way to establish different HCV replicon strains without
the introduction of adaptive mutations. Thus, identifying the physiologically relevant
NS5A kinases (and phosphatases) remains a high priority. Another prediction from
the above working model is that p58, the hyperphosphorylated form of NS5A, is
specifically linked to down-regulation of HCV RNA replication in cell culture. This
prediction is further supported by results from a recent study, in which extensive
mutagenesis analysis was carried out on a region of NS5A presumably involved in
basal- and hyperphosphorylation (Appel et al., 2005). It was found that mutations in
the central serine cluster reduced NS5A hyperphosphorylation and increased HCV
replication. On the other hand, mutations of the C-terminal serine residues decreased
the formation of p56, but did not affect HCV RNA replication significantly. Another
study showed that the expression of a wild-type NS5A protein, or the introduction
of a wild-type NS5A replicon in trans inhibited replication of NS5A-adapted
replicons, in a dominant-negative fashion (Graziani and Paonessa, 2004). These
results indicate that hyperphosphorylated wild-type NS5A may compete with the
adapted-NS5A protein and down-regulate HCV RNA replication.

Despite these exciting results from HCV replicon-based studies, it has been
shown that the adaptive mutations, especially those negatively affecting NS5A
hyperphosphorylation, inhibit HCV replication following infection of chimpanzees
(Bukh et al., 2002). In addition, similar adaptive mutations have not been observed
in HCV patients. In fact, the putative hyperphosphorylation sites of NS5A are
well conserved among different HCV genotypes/isolates from patients. These
observations raised the concern over the physiological relevance of adaptive
mutations in the HCV replicon system. In addition, questions as to whether the
hyper-phosphorylation of NS5A serves additional biological roles during HCV
infection in vivo have arisen. We may speculate that the hyper-phosphorylation of
NS5A serves as a switch point between HCV RNA replication and downstream
steps, such as virus capsid/particle assembly or virus particle maturation and release
and that the hyperphosphorylated form of NS5A may be actively involved in these
downstream events. As previously mentioned, an interaction between NS5A and the

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core protein has been noted (Goh et al., 2001; Shi et al., 2002). This model suggests
that the basal- and hyper-phosphorylation of NS5A are regulated in a temporal
fashion, presumably by different cellular kinases at different steps of the viral
life cycle, to facilitate a complete, productive infection in vivo. With the recently
establishment of bona fide HCV infection system in cell culture (Lindenbach et al.,
2005; Wakita et al., 2005; Zhong et al., 2005) (see Chapter 16), now it is possible to
examine the differential phosphorylation status of NS5A and its role during different
steps of the HCV infection cycle. Additional modes by which NS5A might affect
HCV replication have also been suggested. Most recently, NS5A was shown to bind
with high affinity to the 3' ends of HCV plus- and minus-strand RNAs (Huang et al.,
2005). NS5A might also indirectly regulate HCV replication by modulating HCV
IRES-dependent translation (He et al., 2003; Kalliampakou et al., 2005; Wang et
al., 2003) and its ability to modulate cellular antiviral pathways stimulated by IFNs
has been well documented (Gale and Foy, 2005; Tan and Katze, 2001).

NS5A AS A VIRAL INTERCEPTOR OF CELLULAR PATHWAYS

Interaction with and modulation of host cell signaling pathways constitute an
important aspect of many viral life cycles. HCV is no exception to this, and NS5A
in particular may play a pivotal role in the interaction between HCV and cellular
signal transduction pathways. The interplay between NS5A and the IFN system as
well as the role of NS5A in IFN resistance has generated intense interest and has
been extensively studied (Gale and Foy, 2005; Tan and Katze, 2001). In this section,
we will focus on the affects of NS5A on additional cellular signaling pathways
including those involved in growth, cell-cycle control, apoptosis and cell survival,
and cellular stress responses.

NS5A has been shown to interact with a wide variety of host cell proteins and
thus may modulate numerous diverse signal transduction pathways (Table 1).
Among the cellular signaling pathways affected by the NS5A protein, the best
characterized are those relating to cell proliferation and cell-cycle control, apoptosis
and cell survival, and cellular stress responses. Despite many interesting results and
insightful working models, in only a few cases has the functional relevance in the
context of HCV replication been addressed. Thus, in most cases, the observations
discussed below need further verification in model systems that can better simulate
the HCV life cycle in vivo.

NS5A contains proline-rich PXXP motifs representing binding sites for SH3-
domain containing proteins (Fig. 1). These motifs are frequently present in cellular
signaling molecules (Tan et al., 1999). By testing a panel of SH3 domain-containing
cellular proteins, Tan and colleagues found that NS5A specifically interacted
with Grb2, a cellular adaptor protein involved in the growth factor signaling. The
interaction of Grb2 with NS5A occurred through the C-terminal PXXP motif of

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NS5A (He et al., 2002; Tan et al., 1999). This interaction seemed to be mediated
by the two SH3 domains of Grb2 in a cooperative fashion. Consistent with these
findings, EGF stimulation of cells expressing NS5A showed reduced ERK and p38
MAPK activation, which are downstream signaling events mediated by the Grb2
adaptor protein. In addition, NS5A containing mutations within the C-terminal
proline-rich motif neither interacted with Grb2, nor blocked EGF-stimulated ERK
phosphorylation, supporting the direct connection between NS5A interaction with
Grb2 and its effect on downstream MAPK pathways. The NS5A-Grb2 interaction
and the inhibition of ERK phosphorylation by NS5A were also shown by another
group in various mammalian cell types infected with recombinant HSV-1 viruses
carrying NS5A (Georgopoulou et al., 2003). These studies suggest that NS5A can
disrupt the MAPK mitogenic pathway through direct interaction with Grb2, either
by preventing the recruitment of Grb2 to the upstream receptor complexes, or by
disrupting Grb2 interaction with downstream components of the pathway, such as
Sos. However, the original study found no evidence that NS5A reduced Grb2-Sos
association. More recent studies have found that in HCV replicon cells there was
reduced EGF receptor tyrosine phosphorylation and aberrant recruitment of the
Shc and Grb2 adaptor proteins to the receptor. This correlated with reduced Shc
phosphorylation and Ras activation (Macdonald et al., 2005a). While it is unclear
whether the effects observed in replicon cells were caused by NS5A expression, it
suggests that NS5A may disrupt the association of Grb2 and other adaptor proteins
with the upstream receptor complex, thus blocking downstream Ras-Raf-MAPK
activation at a very early step. The interaction between NS5A and Grb2, and
the precise mechanism by which it blocks the downstream pathway need to be
characterized in greater detail.

Grb2 and the downstream MAPK signaling pathways regulate many cellular
processes such proliferation, gene expression, translational control, to name just a
few. Thus targeting Grb2 and its downstream effectors through NS5A may have a
significant influence on cellular functions and the HCV life cycle. In a follow-up
study, it was found that NS5A inhibited the activity of AP1, a mitogenic and stress-
activated transcription factor, through inhibition of the ERK pathway, and these
effects were dependent upon the C-terminal Class II proline-rich motif that interacts
with Grb2 (Macdonald et al., 2003). It was later shown in another study that an HCV
replicon carrying a mutation within the C-terminal proline-rich motif lost the ability
to block AP1 activation (Macdonald et al., 2005b). These results suggest that NS5A
interaction with Grb2 may affect activation of the MAPK-dependent transcription
factors and thus cellular gene expression. In addition, the ERK and p38 MAPK
pathways also play a role in IFN signaling, by mediating serine phosphorylation
of STAT1/3 transcription factors and contributing to maximal induction of IFN
stimulated genes (He and Katze, 2002). Thus, in addition to its ability to modulate
IFN responses directly by inhibiting the function of PKR, the ability of NS5A to

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 Table 1. Proteins reported to interact/associate with NS5A.
Protein Protein category Interacting NS5A In vivo Biological effect/function of Reference
He et al.

region(s) interaction? interaction

PKR Cellular antiviral kinase PKR-BD (a.a. 237-302) Yes Repression of PKR and Gale et al., 1997; Gale et
downstream pathways al., 1998
CKII Cellular CMCG family N-terminal region? - NS5A phosphorylation? Kim et al., 1999
kinase
Grb2 Cell signaling adaptor C-terminal Class II Yes Disruption of downstream Tan et al., 1999; He et al.,
protein proline-rich motif mitogenic signaling 2002; Georgopoulou et al.,
2003; MacDonald et al.,
2003
hVAP-33/hVAP-A Cellular membrane a.a. 2177-2228 Yes HCV RNA replication complex? Tu et al., 1999; Evans et
protein al., 2004
SRCAP Cellular transcription ? Yes Down-regulation of p21 Ghosh et al., 2000
factor promoter activity

278
karyopherin beta 3 ? ? Yes ? Chung et al., 2000
Cdk1 Cell cycle control protein ? Yes Cell growth and cell cycle Arima et al., 2000
perturbations?
Core HCV capsid structural a.a. 236-354 Yes HCV virus particle assembly? Goh et al., 2001
protein
p53 Cellular transcription ? Yes Inhibition of transcriptional Majumder et al., 2001;
factor transactivation by p53 Lan et al., 2002; Qadri et
al., 2002
ApoA1 Cellular apolipoprotein a.a. 1-224 Yes Colocalization in the Golgi Shi et al., 2002
apparatus?
hTAF(II)32/ Transcriptional ? Yes Inhibition of transcriptional Lan et al., 2002
(TFIID) coactivator transactivation by p53
NS5B HCV polymerase a.a. 105-162 and 277-334 ? Regulation of HCV replication? Shirota et al., 2002;
Shimakami et al., 2004
p85 PI3K Cellular kinase N-terminal region; a.a. Yes Up-regulation of PI3K/AKT He et al., 2002; Street et
271-300 survival pathway al., 2004
 Gab1 Cell signaling scaffold - Yes Indirect association via p85 PI3K He et al., 2002
protein
TBP Cellular transcription Indirect association via Yes Inhibition of transcriptional Qadri et al., 2002
factor p53? transactivation by p53
TRADD Apoptosis signaling Indirect association via Yes Perturbation of TRADD Majumder et al., 2002
adaptor TRAF2? signaling/apoptosis
TRAF2 Cell signaling adaptor Middle one-third (a.a. Yes Perturbation of TFAF2 signaling Park et al., 2002; Park et
protein 148-301) (NFkB, JNK) al., 2003
amphiphysin II Cellular adaptor protein C-terminal proline-rich Yes ? Zech et al., 2003
region
Bax Cellular pro-apoptotic Bcl-2 homology domains Yes Inhibition of apoptosis Chung et al., 2003
protein
La Cellular RNA-binding N-terminal region (a.a. ? ? Houshmand et al., 2003
protein 1-83)
PTX1 Cellular homeodomain ? Yes Perturbation of IFN response? Ghosh et al., 2003

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protein
2-5OAS IFN induced antiviral N-terminal (a.a. 1-148) Yes Perturbation of IFN antiviral Taguchi et al., 2004
protein response
Hck, Lck, Lyn, Fyn Cellular Src family C-terminal Class II Yes ? MacDonald et al., 2004
kinases proline-rich motif
HSP27 Cellular heat shock N-terminal region (a.a. Yes ? Choi et al., 2004
resonse protein 1-181)
Jak1 Cellular IFN signaling ? Yes STAT3 activation Sarcar et al., 2004
kinase
FBL2 Cellular ? Yes HCV replication Wang et al., 2005
geranylgeranylated
protein
HCV NS5A
He et al.

modulate MAPK signaling may also contribute to the ability of HCV to modulate the
IFN response. In addition, NS5A also blocked phosphorylation of eIF4E following
EGF stimulation, which may provide a mechanism for down-regulation of eIF4E-
dependent translation of capped cellular mRNAs, thus favoring cap-independent
translation of HCV RNA (He et al., 2001).

In addition to Grb2, the C-terminal proline-rich motif of NS5A was also found to
mediate interaction with the SH3 domain of several Src kinase family members,
including Hck, Lck, Lyn, and Fyn (Macdonald et al., 2004). NS5A interacted with
these Src family members in vivo and differentially regulated their kinase activity,
inhibiting Hck, Lck, and Lyn while activating Fyn. Similar findings were noted in
HCV replicon cells. However, the downstream effects as well as the physiological
role of the NS5A interaction with these Src kinases remain unclear. It seems quite
remarkable that one particular motif of NS5A is able to interact with so many cell
signaling molecules. Despite all these interesting findings, the physiological role
of the NS5A C-terminal proline-rich motif remains unknown, since mutation of
this motif in an HCV replicon did not affect HCV RNA replication (Macdonald et
al., 2005a). It may be possible that the conserved proline-rich motif is involved in
other steps of the HCV infection life cycle, and this issue might be addressed with
the recently development HCV infection system in cell culture. Alternatively, this
motif and its interaction with cellular proteins might be required for successful HCV
infection and viral pathogenesis in patients, but not for HCV life cycle in tissue
culture, in which many aspects of the in vivo infection are missing.

NS5A has also been shown to interact with and modulate another pivotal cellular
pathway, the PI3K-AKT cell survival pathway (Macdonald et al., 2005a). NS5A
directly interacts with the p85 regulatory subunit of PI3K through the SH3
domain of p85. This interaction may involve either the N-terminal region, or a
novel motif within the middle one-third of NS5A protein. NS5A was found to
bind to heterodimeric PI3K in transient expression systems and enhanced the
phosphotransferase activity of p110, the catalytic subunit of PI3K. The exact
mechanism by which NS5A activates PI3K is not known, but NS5A expression
increased the tyrosine phosphorylation of p85 PI3K following stimulation with
EGF, indicating that NS5A interaction might facilitate the activation of PI3K by
upstream signaling complexes. Along these lines, NS5A and p85 PI3K appeared to
form a complex with Gab1, a cellular docking protein that provides a platform for
the recruitment and activation of downstream signaling molecules in the vicinity
of various growth factor and cytokine receptors (He et al., 2002). Stimulation of
PI3K activity by NS5A results in increased phosphorylation and activation of
AKT/PKB (He et al., 2002; Street et al., 2004). NS5A expression also modulated
serine phosphorylation and function of the proapoptotic protein BAD, also a direct
substrate of AKT. This indicates that NS5A might modulate host cell survival and

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contribute to HCV persistence by interacting with the PI3K-AKT cell survival

pathway. Indeed, NS5A activation of the PI3K-AKT pathway correlated with the
protection against apoptosis in NS5A-expressing cells or HCV replicon cells (Street
et al., 2004). However, NS5A may also disrupt apoptosis through other mechanisms
in these systems (see following parts in this section). In addition, expression of
the HCV polyprotein in cells also activated the PI3K-AKT pathway, resulting in
the modulation of two other AKT substrates, the Forkhead transcription factor
and GSK-3β, indicating that HCV may affect multiple AKT-mediated pathways
and biological functions (Street et al., 2005). Still, the downstream effects of
the interaction between NS5A and the PI3K-AKT pathway are not completely
understood and require clarification. Collectively, results from the studies reviewed
here suggest a multi-faceted model of NS5A action in which NS5A is involved in
the modulation of various cellular pathways. What is not so clear is which of these
cellular pathways are physiologically important during HCV infection in vivo, and
how the interactions between NS5A and multiple signaling pathways are coordinated
and regulated during the HCV replication process.

Previous studies have shown that NS5A promotes cell proliferation resulting
in cellular transformation through a PKR-dependent mechanism (Gale et al.,
1999; Gimenez-Barcons et al., 2005). NS5A may also directly interact with the
cell cycle control machinery. Several studies showed that NS5A repressed the
expression of p21WAF1, a cell cycle regulatory gene (Ghosh et al., 2000b; Ghosh
et al., 1999; Gong et al., 2004; Lan et al., 2002; Majumder et al., 2001; Qadri et
al., 2002) resulting in increased cell proliferation and a transformed phenotype.
The downregulation of p21 expression might involve direct NS5A interaction
with SRCAP, a cellular transcription factor (Ghosh et al., 2000b), and in addition
was suggested to be dependent on the tumor suppressor gene, p53 (Majumder et
al., 2001). NS5A directly bound to and co-localized with p53 in the perinuclear
membrane region, which may cause sequestration of p53 in this region (Lan et al.,
2002; Majumder et al., 2001). NS5A inhibited the transcriptional activation activity
of p53, resulting in inhibition of p21 expression, which is activated by p53 (Lan
et al., 2002; Qadri et al., 2002). NS5A repression of p53 activity might involve
additional factors in the p53 transcriptional activation complex. For example,
NS5A was found to interact and co-localize with hTAF(II)32, a co-activator
of p53. In addition, NS5A formed a heterotrimeric complex with TBP and p53
and inhibited the binding of these two proteins to their consensus DNA binding
sequences (Lan et al., 2002; Qadri et al., 2002). These observations suggest that
NS5A has the potential to interact with multiple cellular transcription factors and
regulate the expression of cell-cycle control genes. However, in contrast to these
experiments, two other studies showed that NS5A expression actually inhibited cell
proliferation in various cell types, which exhibited a reduced S phase and an increase
in the G2/M phase (Arima et al., 2001; Siavoshian et al., 2004). The underlying

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mechanism was suggested to be either p53-dependent induction of p21 (Arima

et al., 2001), or through a p53-independent mechanism (Siavoshian et al., 2004).
The underlying reason for the discrepancy in these results is not clear, but may be
due to the different assay systems being utilized. Overall, the mechanistic details
of how NS5A affects cell cycle control pathways are still not well understood and
await further characterization in the HCV infection system.

Apoptosis is a proactive cell death process and in some cases is caused by viral
infection. Prevention of host cell apoptosis may be beneficial to viruses by
allowing longer periods of viral replication and persistence. It has been shown that
NS5A could disrupt the apoptotic process through either PKR- or p53-dependent
mechanisms (Gale et al., 1999; Lan et al., 2002). In addition, NS5A was able to
inhibit apoptosis induced by treatment of human hepatoma cell lines with TNF-
α. This effect correlated with a block in the activation of cellular caspases and
downstream proapoptotic events (Ghosh et al., 2000a; Miyasaka et al., 2003).
Interestingly, in transgenic mice expressing NS5A in the liver, TNF-induced
apoptosis was prevented (Majumder et al., 2002). NS5A was found to physically
associate with the TRADD signaling complex, which associates with the TNF
receptor, and reduced the interaction between TRADD and FADD (Majumder et al.,
2002; Park et al., 2002). So it seems that NS5A can block TNF-dependent apoptosis
by associating with and disrupting the TRADD-FADD signaling complex.

In addition, it was found that NS5A expression inhibited TNF-induced activation

of NK-κB, which is mediated by TRADD and TRAF2 (Majumder et al., 2002;
Park et al., 2002). Consistently, NS5A directly interacts with and co-localized
with TRAF2. The interaction was mapped to a.a. 148-301 of NS5A and required
the TRAF-domain of TRAF2. However, NS5A did not block the recruitment of
either TRAF2 or IKK-β to the TNF receptor complex, suggesting that NS5A may
form a multi-subunit complex with at least TRAF2 and TRADD in the vicinity
of TNF receptor (Park et al., 2003). Curiously, NS5A was also found to enhance
TRAF2-mediated JNK activation by TNF-α (Park et al., 2003). It is unclear
how the NS5A-TRAF2 interaction differentially modulates the NF-κB and JNK
pathways. However, it is tempting to speculate that NS5A might disrupt host cell
inflammatory and immune responses. What affect this might have in the context
of HCV infection is not known. In addition, whether or not the NS5A-TRAF2
interaction is required for HCV RNA replication in cell culture requires further
testing. Given that TRAF2 may also mediate cellular ER stress response and PKR-
dependent NF-κB activation, it is possible that NS5A interaction with TRAF2 may
also affect these cellular processes as well.

In another study, NS5A was able to antagonize sodium phenylbutyrate (NaPB)-

induced apoptosis in hepatocellular carcinoma cells, a p53-independent process

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(Chung et al., 2003). NS5A was shown to co-localize and interact with Bax, a pro-
apoptotic Bcl-2 family member, in the nucleus after NaPB treatment. Surprisingly,
NS5A was found to contain a few Bcl-2 homology domains (BH3, BH1, and BH2;
Fig. 1), which are domains found in Bcl-2 family members and mediate interaction
between Bcl-2 proteins. BH3 and BH1 are in the N-terminal half of NS5A, while
BH2 partially overlaps with the ISDR. A mutant of NS5A deleted for both BH2 and
the putative NLS regions localized to the cytoplasm and disrupted its association
with Bax. In addition, this mutant protein was no longer able to suppress NaPB-
induced apoptosis. On the other hand, deletion of the NLS region alone resulted
in a protein which still associated with Bax in the perinuclear region, but showed
reduced association with Bax in the nucleus and reduced ability to block NaPB-
induced apoptosis. These results suggest that NS5A may act as a Bcl-2 analogue
and interact with Bcl-2 family members to block the apoptosis pathway, and this
process may require the nuclear form of NS5A protein. As discussed previously,
the biological function of the nuclear form of NS5A and the mechanism by which
it is produced is not clear. Similarly, the role of the nuclear form of NS5A as it
pertains to HCV infection requires additional experimentation.

Viral infection frequently results in activation of host cell defense mechanisms and
stress responses, due to overexpression of viral proteins, stimulation of the innate
immune responses pathways such as the IFN system, and disruption of normal
cellular functions. In contrast to the above mentioned studies on the TNF receptor,
Gong and colleagues found that NS5A expression activated the NF-κB and STAT3
transcription factors through oxidative or ER stress (Gong et al., 2001; Waris et al.,
2002). NS5A seems to trigger oxidative stress by disturbing intracellular calcium
pools, and the activation of NF-κB and STAT3 by NS5A is sensitive to inhibition
by antioxidants and calcium chelators. Activation of the NF-κB pathway was also
confirmed by microarray analysis of Huh7 cells expressing NS5A, since many
NF-κB responsive genes were identified (Girard et al., 2004). The activation
of the NF-κB pathway by NS5A may involve a novel mechanism involving
tyrosine phosphorylation of IκB-α at two sites (Tyr42 and Tyr305) suggesting an
alternative activation mechanism (Waris et al., 2003). Additionally, oxidative stress
and activation of NF-κB have also been observed in HCV replicon cells, but it is
unclear whether these effects are specifically caused by NS5A (Qadri et al., 2004;
Waris et al., 2003). In addition, in NS5A-expressing transgenic mice, activation
of the STAT3 transcription factor was also observed in the mouse liver (Sarcar et
al., 2004). In this study, it was suggested that the activation of STAT3 by NS5A
might involve the association of NS5A with the Jak1 kinase. It is unclear whether
the NS5A-Jak1 association occurs during IFN signaling or whether this has an
impact on IFN-induced antiviral responses. It is noteworthy that many of the cell
culture-based studies reviewed in this chapter involve overexpression of NS5A
and other HCV proteins at concentrations that are most likely higher than those in

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HCV-infected liver cells in patients. Thus, all these studies need to be considered in
the proper context. Thus, future studies will more than likely be aimed at verifying
these results in experimental systems that are more physiologically relevant as they
become available.

In addition to the cellular signaling pathways discussed above, NS5A has also
been reported to interact with a wide variety of cellular proteins. These NS5A-
interacting proteins include karyopherin beta 3 (Chung et al., 2000), the adaptor
protein amphiphysin II (Zech et al., 2003), the homeodomain protein PTX1 (Ghosh
et al., 2003), and HSP27 (Choi et al., 2004), among many others [Table 1]. The
exact physiological affects of these interactions require follow-up studies, but it
seems likely that the range of cellular signaling pathways that are affected by NS5A
is likely to expand.

CONCLUDING REMARKS
Tremendous progress has been made in our understanding of the biology of the NS5A
protein. Recent biochemical and structural studies have given us great insight into
the location of NS5A in various cellular compartments and the domain architecture
of this protein. Various cellular binding partners have been identified and the affects
of NS5A on various cellular signal transduction pathways continue to be an area of
great interest. The role of phosphorylation of NS5A by host cell kinases continues
to be defined. In addition, the advancement of the HCV replicon system has shed
light on the physiological role of NS5A in viral replication. Despite this progress,
several key questions remain. The precise role of the various forms of NS5A both
in terms of subcellular localization and phosphorylation needs be systematically
addressed. In addition, the role of other NS proteins in phosphorylation needs to
be further refined and the clear identification of host cell kinases leading to both
basal and hyperphosphorylation of NS5A requires additional work. One of the
most exciting areas of research on NS5A is its interactions with host cell proteins
and its ability to modulate host pathways. In most cases, the physiological role of
these interactions needs to be studied in the context of viral replication. While we
have learned much about the ability of NS5A to modulate the IFN response, more
research is needed into its effects on other aspects of innate immunity. With the
recent development of an HCV infection model, future work will most certainly
be aimed at investigating the role of NS5A in other aspects of the HCV life cycle
including viral entry and assembly. As new in vivo models evolve (see Chapter 12),
the role of NS5A in virus replication in animal models will certainly be defined.
Thus, future research should provide new clues as to the various functions of this
truly remarkable multifunctional regulator.

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Chapter 10

Biochemical Activities of the HCV NS5B

RNA-Dependent RNA Polymerase
C. T. Ranjith-Kumar and C. Cheng Kao

ABSTRACT
Structural and functional studies of the hepatitis C virus (HCV) RNA-dependent
RNA polymerase have contributed to our understanding of polymerase mechanism,
viral RNA replication, and have generated targets for antiviral development. This
review summarizes recent studies on the properties of the HCV polymerase.

INTRODUCTION
HCV, like other (+)-strand RNA viruses, uses its viral genomic RNA as a template for
both translation and generation of a complementary (-)-stranded RNA intermediate.
The (-)-stranded RNA is then used as the template for the synthesis of molar excess
of (+)-stranded progeny RNA molecules. (For a good general review on RNA virus
replication, see Buck, 1996.) A membrane-associated replicase enzyme complex
consisting of virally encoded and host proteins is responsible for the replication
of viral RNA. The catalytic subunit of the replicase complex is the HCV encoded
nonstructural 5B protein (NS5B), which contains all the sequence motifs highly
conserved among all the known RNA-dependent RNA polymerases (RdRps) (Poch
et al., 1989). By extension of studies from the human immunodeficiency virus
(HIV), where the reverse transcriptase is a primary target for effective antivirals,
the HCV RdRp is considered an important target for drug development (Beaulieu
and Tsantrizoa, 2004; Wu and Hong, 2003).

Analysis of HCV replication has been hampered by the lack of convenient animal
model and efficient cell culture systems. As a result, compounds against HCV
have been screened using either surrogate viruses such as bovine viral diarrhea
virus (BVDV) (Bukhtiyarova et al., 2001), biochemical targets such as the NS3
protease-helicase and/or the NS5B RdRp (Sarisky, 2004), and hepatoma cell line
Huh7 expressing the subgenomic replicon (Lohmann et al., 1999; Blight et al., 2000;
Guo et al., 2001; Ikeda et al., 2002). Subgenomic replicons are increasingly used
to screen and characterize antivirals (Horscroft et al., 2005), although inhibitors
identified using such cell-based screens still need to be tested against all viral
proteins encoded by the replicon system to determine the mechanism of action. Thus,
the HCV NS5B remains a target of choice for both nucleoside and nonnucleoside

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Fig. 1. A schematic of the HCV RdRp depicting the locations of the motifs and domains. The sequence
alignments of the six recognizable motifs within RdRps from HCV, the double-stranded RNA phage
φ6 and poliovirus 3Dpol are also shown.

inhibitors. This chapter we will focus on the structural and functional aspects of
the HCV RdRp.

EXPRESSION OF NS5B
Expression of recombinant NS5B in insect and bacterial cells provided valuable
reagents for the biochemical characterization. NS5B expressed using the
baculovirus system can perform RNA-dependent RNA synthesis (Behrens et al.,
1996; Lohmann et al., 1997). However, generation of soluble full length NS5B in
bacterial cells proved unsuccessful in spite of a number of attempts (Yuan et al.,
1997). A hydrophobic profile of the NS5B revealed that the C-terminal 21 amino
acid residues is highly hydrophobic and is predicted to insert into membrane. In
fact, membrane association of the RdRp is essential for the replication of HCV
subgenomic replicons in cells (Moradpour et al., 2004). Deletion of this C-terminal
tail of NS5B resulted in a soluble protein that had properties similar to that of
protein expressed using insect cells, indicating that the C-terminal tail contributes
minimally to nucleotide polymerization (Yamashita et al., 1998). Vo et al. (2004)
have evidence suggesting that the C-terminal tail in the recombinant NS5B protein
will increase interaction with RNA. However, it is unclear how a hydrophobic
region can affect RNA binding.

Enzymatically active NS5B proteins fused to glutathione S-transferase or a histidine

tag have been generated (Yamashita et al., 1998; Oh et al., 1999; Ferrari et al., 1999).
However, NS5B with a N-terminal histidine tag was expressed at a lower level and
also had lower activity compared to the C-terminally tagged protein (Lohmann
et al., 1997; Ferrari et al., 1999). This phenomenon may be related to the results
from Lohmann et al. (1997, and 1998), who found that changes in the N-terminal
residues of NS5B reduced RdRp activity in vitro (Lohmann et al., 1997 and 1998)
and that residue Cys14 of NS5B contributes to RNA binding (Bressanneli et al.,
1999; O'Farrell et al., 2003).

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Fig. 2. The structure of the HCV RdRp. The figure is from Lesburg et al. (1999), reprinted with
the permission of the publisher and modified to denote the positions of the thumb, finger and palm
domains, and the location of the template channel. The colored structures denote conserved motifs
within the RdRp: red, motif A; green, motif B; yellow motif C; light violet,motif D; purple: motif E;
dark grey, motif F. The β-loop is in light brown.
A colour version of this figure is printed in the colour plate section at the back of this book.

STRUCTURAL FEATURES OF HCV NS5B

Similar to other known RdRps, the HCV NS5B also contains six conserved motifs
designated A-F. Comparison of the HCV NS5B sequence with the poliovirus
RdRp and the φ6 RNA polymerase, two other model RdRps, is shown in Fig.
1. The three-dimensional crystal structure of HCV NS5B has been determined
independently by several groups (Lesburg et al., 1999; Bressanelli et al., 1999;
Ago et al., 1999). Using the right-hand analogy for polymerases (Joyce and Steitz,
1995), the HCV RdRp has discernable fingers, palm and thumb subdomains. An
unusual feature of this polymerase is that, due to the extensive interactions between
the finger and thumb subdomains, the HCV RdRp has an encircled active site (Fig.
2) (Lesburg et al., 1999; Bressanelli et al., 1999; Ago et al., 1999). These contacts
restrict the flexibility of the subdomains and possibly constrain flexibility between
the subdomains. The HIV reverse transcriptase and other DdRps are known to
undergo transition from an open or to closed conformation upon template binding
and polymerization (Doublie et al., 1999; Huang et al., 1998). Structural analysis
of HCV NS5B (J4 strain) revealed that de novo initiation is the probable mode of
RNA synthesis, and limited structural changes take place upon nucleotide binding

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Fig. 3. The catalytic NTP binding pocket in the HCV RdRp. The structure is from Bressanelli et al.
(2002) and is reprinted with the permission of the publisher. Left panel: a detailed view of the NTP
binding site in complex with rUTP. Amino acids involved in binding NTP are labeled. Divalent metals
are depicted as grey spheres labeled with A and B. Right panel: a detailed view of the active site of
the HCV RdRp with the potential entry portal for incoming NTPs.
A colour version of this figure is printed in the colour plate section at the back of this book.

(O'Farrell et al., 2003). Structural studies with RdRp from the HCV genotype 2a
indicate the presence of two conformations of the protein even in the absence of
template RNA, where the key difference between the two forms is the relative
orientation of the thumb domain in relation to fingers and palm domains (Biswal
et al., 2005). Since both conformations lacked RNA, whether the template RNA
will induce the same structural change(s) remains to be determined.

Another unusual feature of NS5B is a β-hairpin loop that protrudes into the active
site located at the base of the palm subdomain (Fig. 2). This 12 amino acid loop
was suggested to interfere with binding to double stranded RNA due to steric
hindrance (Hong et al., 2001). The poliovirus RdRp lacks a similar β-loop, a feature
that may be related to the poliovirus RdRp normally directing genome replication
with a protein-nucleotide primer (Paul et al., 1998). It was suggested that the β-
loop could be involved in positioning the 3' terminus of the viral RNA for correct
initiation (Hong et al., 2001). Since the wild-type HCV RdRp is fully capable of
primer-dependent RNA synthesis, additional factors are needed to prevent primer
extension. Indeed, GTP can, along with structures within the RdRp, help prevent
primer-extension (Ranjith-Kumar et al., 2003).

The C-terminal tail of NS5B also lines the RNA binding cleft in the active site. This
region, which immediately precedes the C-terminal membrane anchorage domain,
forms a hydrophobic pocket and interacts extensively with several important
structural elements including the β-loop (Adachi et al., 2002; Leveque et al., 2003).

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Fig. 4. A low affinity rGTP binding site in the HCV RdRp. The structure is from Bressanelli et al.
(2002) and reprinted with the permission of the publisher. Left panel: a view of the back of the HCV
RdRp emphasizing the low affinity GTP binding pocket. The arrows denote β-strands, the cylinders
denote helices and lines denote connecting loops. The low affinity GTP binding site resides in the
blue colored region containing the thumb subdomain. Right panel: a more detailed view of the low
affinity rGTP binding pocket.
A colour version of this figure is printed in the colour plate section at the back of this book.

Deletions of up to 55 residues of the C-terminal tail resulted in increased RdRp

activity, suggesting a direct role for the C-terminal tail in RNA synthesis activity
and providing evidence for regulation at the active site (Lohmann et al., 1997;
Tomei et al., 2000; Ranjith-Kumar et al., 2002).

CATALYTIC POCKET
The active sites of HCV NS5B and HIV-1 reverse transcriptase are very similar and
can be superimposed without significant steric clashes (Lesburg et al., 1999). The
residues involved in nucleotidyl transfer are found in palm motifs A and C. Motif
A harbors the metal binding residue D220 which is a part of conserved D-X4-D
motif, while motif C has the conserved metal binding and nucleotidyl transfer
residues D318 and D319 (Fig. 3). D225 within motif A forms a H-bond with the
ribose 2'-hydroxyl group of the NTP and is thought to discriminate against the use of
dNTPs. Crystal soaking experiments with HCV NS5B with NTPs revealed several
residues in the catalytic pocket that contact the triphosphates of NTP (Fig. 3). These
include R158, S367, R386, T390 and R394. Unfortunately, it was not possible to
identify the base-interacting residues. The role of these residues in RdRp activity
is discussed in more detail later in this chapter.

Soaked NS5B crystals with GTP also revealed a low affinity GTP binding site on
the surface of the protein (Fig. 4; Bressanelli et al., 2002). This second site, which
is apparently specific for GTP, lies between the fingers and thumb subdomains,
approximately 30 Å away from the catalytic pocket. By virtue of its position and the

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requirement of higher GTP concentrations to saturate the pocket, it was suggested

that it might play a regulatory role in RNA synthesis (Bressanelli et al., 2002). To
date, any allosteric properties of this low affinity site in response to GTP has not
been determined.

ACTIVITIES OF NS5B
Recombinant NS5B was sufficient to synthesize full length HCV RNA in vitro
(Hwang et al., 1997; Yamashita et al., 1998; Behrens et al., 1996; Lohmann et
al., 1997; Ferrari et al., 1999). This observation indicates that NS5B can also
unwind stable secondary and tertiary RNA structures. As covered in Chapter 2 of
this book, the 5' and 3' untranslated regions (UTRs) of the HCV genome contain
highly ordered and complex RNA structures , which are highly conserved and
contain cis-acting elements for viral RNA replication. Recently it was shown that a
pseudoknot structure is formed between the 3' end of the HCV genome and a novel
RNA element in the NS5B coding sequence (Friebe et al., 2005). The 3' terminal
150 nt of the HCV RNA contain signals that are essential for RdRp binding and
replication of viral RNA (Yi and Lemon, 2003a and b; Reigadas et al., 2001; Cheng
et al., 1999). In vitro HCV NS5B was found to replicate the 3' terminal region
of the (-)-strand RNA more efficiently than the 3' terminal region of (+)-strand
RNA (Reigadas et al., 2001). Analysis of the promoter elements in 3' terminus of
(-)-strand revealed that complementary strand of second stem-loop of the internal
ribosome entry sequence (IRES) binds NS5B and acts as a positive element for
RNA synthesis (Kashiwagi et al., 2002). However, the complementary strand of
the first stem-loop of the IRES worked as a negative regulator of RNA synthesis
(Kashiwagi et al., 2002).

Though some genome specific recognition was observed, recombinant NS5B largely
lacked specificity for binding to HCV RNA (Lohmann et al., 1997). Several groups
used different RNAs to analyze RdRp activity. In general, the HCV NS5B preferred
a template with a stable 5' secondary structure(s) and a single stranded sequence that
contained at least one 3' cytidylate (Kao et al., 2000). This observation was further
supported when high-affinity RNA ligands to HCV NS5B were isolated using the
Systematic Evolution of Ligands by EXponential enrichment procedure (Vo et al.,
2003). The high affinity RNA was found to have three stem-loop structures.

Our lab uses short RNAs to study de novo initiation of RNA synthesis by the HCV
NS5B because products from these templates can be identified with single-nucleotide
resolution. The prototype RNA, LE19, was derived from BVDV. LE19 is predicted
by mfold to form a stem-loop with five intramolecular base pairs and with single-
stranded sequences of three nucleotides at both the 5' and 3' ends (Fig. 5). The 3'
sequence contains a cytidylate that can be used as an initiation nucleotide. Two LE19
molecules also form a heterodimer which can be extended to form a 32-nt primer

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extension product. In addition, NS5B can add nontemplated nucleotides to the RNA,
a process known as terminal nucleotidyl transferase (TNTase). Lastly, HCV RdRp
can generate recombinant RNA product from two or more non-covalently linked
templates, a process known as template switch. All these activities can be studied
using LE19 in a single reaction (Fig. 5). The different activities of NS5B and their
potential importance in viral replication are discussed in detail below.

DE NOVO SYNTHESIS AND PRIMER EXTENSION

Initiation of RNA synthesis in infected cells likely starts de novo, by use of a one-
nucleotide primer (Bressanelli et al., 2002; Ferrari et al., 1999). This process is well
studied in DNA-dependent RNA polymerase, which uses two NTP binding sites in

Fig. 5. An RNA template that can be used to examine several activities of the HCV RdRp in one
reaction. A) Schematics of the various activities of the HCV RdRp using LE19. LE19 exists in an
equilibrium between monomeric and dimeric forms. De novo initiation occurs at the 3'- terminal
cytidylate of the monomer to generate a 19-nt product. Two LE19 molecules could base-pair through
the nucleotides at their 3'-termini to generate templates for primer extension. In addition, de novo
initiation from one template could result in a ternary complex that does not terminate, but instead
uses a second template to form a template switch product. Lastly, the 3' terminus of LE19 could act
as the acceptor for nontemplated nucleotide addition. B) A demonstration of the effects of GTP and
Mg2+ on the activities of the HCV RdRp. The autoradiogram shows the products synthesized by the
HCV RdRp Δ21 or Δ21 with mutations in the divalent metal-binding residues (D318 and D319).
The presence or absence of GTP are noted with "+" and "-", respectively. The length of the RNAs
(in nucleotides), and the mechanism used to generate a product are noted to the right and left of the
autoradiogram, respectively.

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the catalytic pocket. The first site is called the I site and specifically recognizes the
initiating NTP (NTPi). The second site, the I+1 site, is less specific and recognizes
the NTP complementary to the second template nucleotide. The flaviviral RdRps can
also accommodate two NTPs, with the I site preferentially binding to GTP (Ferrari
et al., 1999; Kao et al., 1999; Lohmann et al., 1999; O'Farrell et al., 2003; Oh et
al., 1999; Luo et al., 2000; Ranjith-Kumar et al., 2002; Sun et al., 2000; Zhong et
al., 2000; Zhong et al., 2000). The catalytic aspartates coordinate divalent metals
that are in position to help form a phosphodiester bond between the NTPi and the
second NTP (Ferrari et al., 1999; O'Farrell et al., 2003).

While GTP is generally accepted as the NTPi for RNA synthesis by the HCV RdRp
in vitro, the 3' terminal residue of many HCV isolates is a uridylate, suggesting
that ATP may be used to initiate (-)-strand RNA synthesis. Recently, a subgenomic
replicon that was passaged in vitro was demonstrated to switch from using GTP
to ATP as the NTPi for both (+)- and (-)-strand RNA replication (Cai et al., 2004).
The exact identity of the NTPi is specific for a purine triphosphate, but can be
somewhat flexible, as it is for DNA-dependent RNA polymerases (Kuzmine et al.,
2003 and references within).

Polymerases require divalent metal ions for activity. RNA synthesis by NS5B is
increased by 4-20 fold when Mn2+ is present in the reaction in comparison to a
reaction with only Mg2+ (Zhong et al., 2000; Ferrari et al., 1999; Luo et al., 2000;
Ranjith-Kumar et al., 2002). However, other divalent metal ions such as Co2+, Cu2+,
Ni2+ and Zn2+ did not support RdRp activity (Luo et al., 2000; Ranjith-Kumar et
al., 2002). Recently it was shown that iron binds specifically to the Mg2+ binding
site of NS5B and can inhibit RNA synthesis (Fillebeen et al., 2005). Mn2+ appears
to more specifically contribute to de novo initiation by lowering the KM for GTP by
about 30-fold (Ranjith-Kumar et al., 2002). While the concentration of Mn2+ used
in vitro is far higher than concentrations present in the cell, physiologically relevant
Mn2+ levels can increase de novo initiation with GTP (Ranjith-Kumar et al., 2002).
Analysis of proteins with C-terminal deletions revealed that the C-terminus of the
HCV RdRp plays a role in Mn2+ induced de novo initiation and can contribute to
the suppression of primer extension (Ranjith-Kumar et al., 2002). Spectroscopy
examining the intrinsic tryptophan and tyrosine fluorescence of the HCV RdRp
produced results consistent with the protein undergoing a conformational change in
the presence of divalent metals (Ranjith-Kumar et al., 2002; Bougie et al., 2003).

Even though de novo initiation seems to be the preferred mechanism of initiation,

it has always been puzzling that primer-extension activity was far more robust than
de novo initiation in vitro. This begs the question of whether the two mechanisms
will affect each other. Deletion of the β-loop led to an increase in primer extension
activity (Hong et al., 2001). However, this deletion was only part of the requirement

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for the suppression of primer extension since additional characterizations revealed

that the NTPi-binding site, higher concentrations of the initiation GTP, and the
C-terminal tail that lines the catalytic pocket all participate to suppress primer
extension (Ranjith-Kumar et al., 2003). Thus, the features and requirements for de
novo initiation by the HCV RdRp significantly prevent primer extension.

MUTATIONAL ANALYSIS
Site directed mutagenesis has been employed to study the role of specific residues
in RdRp activities (Lohmann et al., 1997, Qin et al., 2001; Labonte et al., 2002;
Ranjith-Kumar et al., 2002, 2003 and 2004). Mutation of the conserved catalytic
residues D318 and D220 resulted in inactive proteins. Lohmann et al. (1997)
investigated the effects of mutations of some conserved residues in motifs A, B, C
and D. Most of the mutations on the conserved residues in motifs A and B led to
inactive protein and inactive subgenomic replicons in cell culture (Cheney et al.,
2002). However, mutations of the conserved residues G317, and D319 in motif C
were tolerated somewhat. Interestingly, substitution of R345 with lysine in motif
D enhanced the enzymatic activity (Lohmann et al., 1997). A similar increase in
activity was observed when K151 was mutated to glutamate (Labonte et al., 2002).
Qin et al., (2001) generated a series of clustered and point mutations and studied
their effects on RdRp activity and template binding. The residues that affected
RdRp activity included E18, Y191, C274, Y276 and H502. Y276 was also found
to be important for interaction with the template/primer.

The structures of the HCV RdRp and nucleotides identified a number of interacting
residues at D225, R48, R158, R386, R394, and S367 that interact with the initiation
GTP (Bressanelli et al., 2002; Fig. 3). In this structure, it is not clear whether the
GTP is binding to the I site or the I+1 site. NTPi binding to the I site is base-
specific while binding of the second NTP to the I+1 site should be directed by
the template (Ranjith-Kumar et al., 2002). Because the RdRp-GTP structure was
determined without a template, it is likely that the residues identified recognized
the NTPi. Alanine substitutions of these residues were analyzed for effects on de
novo initiation, primer extension, TNTase, and template switch (Ranjith-Kumar
et al., 2004). Although all mutations retained the capability for primer extension,
alanine substitutions at R48, R158, R386, R394, and D225 decreased de novo
initiation, and two or more mutations in combination abolished de novo initiation
(Ranjith-Kumar et al., 2004). It is likely that these mutations affected the stability
of the initiation complex, since many of the defects were rescued when the reactions
were supplemented with Mn2+. We note that several of the mutant enzymes were
selectively affected for de novo initiation and/or terminal nucleotide addition,
indicating that the residues in the active site can contribute differentially to the
known activities of the HCV RdRp. Furthermore, while the prototype enzyme
had a KM for GTP of 3.5 μM, all mutations except one negatively affected the KM

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for GTP by 3 to 7 fold, demonstrating that the affected residues are functionally
required to interact with the initiation nucleotide. Lastly, mutations in D225 are
dramatically affected in template switch, suggesting that this residue of the NTPi
pocket also participates in the elongation complex.

TERMINAL NUCLEOTIDYL TRANSFERASE (TNTase) ACTIVITY

TNTase activity could be a significant concern in biochemical screens where the
products of the HCV RdRp are not visualized by denaturing gel electrophoresis.
This activity was observed by some (Behrens et al., 1996; Ishii et al., 1999; Ranjith-
Kumar et al., 2001), but not by others, leading some to suggest that it should be
attributed to cellular contaminants rather than the HCV RdRp (Lohmann et al.,
1997, Oh et al., 1999; Johnson et al., 2000; Zhong et al., 2000). Ranjith-Kumar et
al. (2001; 2003) demonstrated that the HCV NS5B does have TNTase activity. First,
mutation of the conserved GDD motif inactivated both polymerase and TNTase
activities. Second, a HCV NS5B specific inhibitor inhibited both activities. Third,
proteins purified from eukaryotic and prokaryotic expression systems both showed
TNTase activity. Fourth, several mutations in the residues in the HCV RdRp that
affected de novo initiation also affected TNTase activity, implicating the NTPi
pocket in TNTase activity. Consistent with this last claim, Mn2+, which increases de
novo initiation, also increases TNTase activity (Ranjith-Kumar, 2004). Perhaps one
reason for the discrepant observations of TNTase activity from different laboratories
is that the amount of TNTase depends on the template sequence and the NTPs used
(Ranjith-Kumar et al., 2001).

TEMPLATE SWITCH
RNA recombination contributes to genetic diversity and pathogenesis of RNA
viruses (Nagy and Simon, 1997; Jarvis and Kirkegaard, 1991). HCV NS5B can
generate RNA products that are larger than the size of the template RNA by a
process wherein the RdRp ternary complex does not terminate RNA synthesis
from a template, but will bind to a second template and continue RNA synthesis.
Template switch by the related BVDV RdRp and replicase complexes from plant
viruses have been characterized and require the template initiation cytidylate as
well as the NTPi (Kim et al., 2001).

Several of the mutations in the NTPi pocket of the HCV RdRp affected template
switch (Ranjith-Kumar et al., 2004). A defect in the template switch is to be expected
with many of the NTPi mutants since fewer ternary complexes are available.
However, mutant D225A, which was capable of robust de novo initiation from the
first template, was debilitated for template switch in comparison to the wild-type
RdRp (Ranjith-Kumar et al., 2004). We hypothesize that D225, which recognizes
the ribose 2' hydroxyl of the NTPi, also plays an additional role either in recognition
of the second template or in the release of the nascent RNA.

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INTERACTION WITH OTHER HCV NONSTRUCTURAL

PROTEINS
HCV replication uses a replicase complex that is associated with the endoplasmic
reticulum (ER) (Hwang et al., 1997). The complex is thought to contain both host and
virally encoded proteins, although detailed information concerning the composition
of the replicase remains to be determined. A growing number of cellular proteins
that could affect HCV replication activity have been identified. For example, it was
recently shown that NS5B is phosphorylated by a protein kinase C-related kinase,
PRK2, and this phosphorylation is involved in regulating HCV replication (Kim
et al., 2004). (For general reviews of cellular factors involved in (+)-strand RNA
virus infection, please see Lai et al., 1998, and Kushner et al., 2003).

HCV nonstructural proteins are co-localized with NS5B within the ER membrane
and likely modulate NS5B activities (Behrens et al., 1996; Brass et al., 2002;
Egger et al., 2002; Hwang et al., 1997; Wolk et al., 2000). One case is that the
HCV NS5B can form oligomers in vitro and may catalyze RNA synthesis in a
cooperative manner (Wang et al., 2002; Qin et al., 2002). While the C-terminal tail
is not involved in this process, it was shown that two amino acids, E18 and H502,
are very critical for oligomerization (Qin et al., 2002). While NS5B oligomerization
can be demonstrated by several independent assays, its role in the infected cell
remains to be determined.

Other interactions between HCV NS5B and other HCV nonstructural proteins
have already been reported with low-resolution assays, such as protein pull-down
experiments, co-immunolocalization, and yeast two-hybrid experiments. These
results indicate that NS5B likely binds to NS3, and that NS3 will interact with
NS4A and possibly NS4B and NS5B (Piccininni et al., 2002). NS5A may interact
with NS2, NS3, NS4A, NS4B, NS5B and with itself (Dimitrova et al., 2003,
Shirota et al., 2002). The NS5A protein is important for HCV replication and
mutation that can increase the efficiency of subgenomic replicon replication can
be mapped to it (Blight et al., 2000; Krieger et al., 2001). By using GST pull down
and coimmunoprecipitation assays, NS5A was shown to directly interact with NS5B
and modulate its activity (Shirotta et al., 2002).

NS3 is a multifunctional protein possessing protease, helicase, and NTPase

activities (Borowski et al., 2002). Recently, the NS5B protein was shown to bind
to NS3 through the protease domain and increase the helicase activity of NS3 by
approximately five fold (Zhang et al., 2005). These findings suggest that HCV RdRp
regulates the functions of NS3 during HCV replication. In contrast, full-length NS3
reduced RNA synthesis by the NS5B RdRp in vitro, possibly due to the NTPase
activity of NS3 degrading NTPs nonspecifically. Whether this effect is relevant in
vivo is unclear, and additional regulations of NS5B activity in the context of the
HCV replicase remain to be characterized.

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A major hurdle in understanding the HCV replication complex is that the replicase
is not only present in relatively low abundance, but enriched replicase can only
perform elongative RNA synthesis from the template RNA that copurified with
the enzymatic activity (Hardy et al., 2003; Lai et al., 2003, Ali et al., 2002). These
features have made the replicase a poor reagent to understand the requirements
for HCV replication in vitro. Nonetheless, the recent demonstration of the ability
to produce HCV elongation-competent replicase is an important first step in its
characterization and could provide a useful reagent to characterize drugs identified
in cell-based screens.

CONCLUSION AND FUTURE TRENDS

Although the initial effort to characterize the HCV RdRp was in response to a need
to develop biochemical targets against HCV, recent research by the virology and
structural biology communities has developed a strong basic understanding of the
mechanism of action of a viral RNA-dependent RNA synthesis. This information
should also prove useful in understanding the mechanism of action of antivirals
targeted against HCV replication. Despite the progress made, much remains to be
done. One bottom line is that we still do not have an effective drug targeting the
HCV RdRp. In terms of future biochemical analyses, we need to: 1) gain a deeper
understanding of the mechanism of polymerization and the conformational changes
as the polymerase goes from initiation to elongative synthesis to the termination of
RNA synthesis. Toward this effort, a true ternary complex at high resolution will be
critical; and 2) define the effects of other viral and cellular subunits that participate
in RNA-dependent RNA replication. Since HCV replication is a process intimately
associated with the cellular membranes, understanding HCV replication will benefit
from an infusion of expertise from membrane biologists and biochemists, and
scientists trained to study the mechanism of complex enzymes, including those who
have analyzed nucleic acid synthesis from other template-dependent polymerases.
It is such multidisciplinary approach that could ultimately yield significant benefits
to society against a disease with enormous worldwide impact.

ACKNOWLEDGEMENTS
We thank our colleagues at Texas A and M University for helpful discussions and Y.
Kim for editing this manuscript. The Kao lab is supported by the National Science
Foundation MCB grant 0332259.

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Chapter 11

HCV Replicon Systems

Keril J. Blight and Elizabeth A. Norgard

ABSTRACT
With the remarkable ability of hepatitis C virus (HCV) to establish persistent
infections that can lead to progressive liver pathology and the poor response of
prevalent HCV genotypes to the current treatment, HCV represents a significant
global health problem. Studies of HCV replication in cell culture were virtually
impossible until the development of subgenomic replicons that replicate
autonomously in the human hepatoma cell line Huh-7. Many improvements
to the replicon system have been made allowing the establishment of transient
replication assays for HCV genotypes 1a, 1b, and 2a. Specifically, the identification
of adaptive mutations that drastically enhance HCV genotype 1 replication and the
isolation of highly permissive Huh-7 sublines led to the development of replication-
competent full-length genomes in addition to a collection of robustly replicating
subgenomes derived from genotype 1 sequences. More recently, the cell tropism
of HCV subgenomic replicons has been expanded to non-hepatoma cell lines and
mouse hepatocytes. The HCV replicon system has opened new avenues for detailed
molecular studies of RNA replication and HCV-host interactions as well as the
development of active inhibitors of HCV replication. Finally, the identification
of genotype 2a-derived replicons that efficiently replicate in cell culture without
adaptive mutations has facilitated the development of systems supporting the
complete virus life cycle.

INTRODUCTION
Persistent infection with HCV has emerged as one of the primary causes of chronic
liver disease, with an estimated 170 million carriers throughout the world (WHO,
2000). Viral persistence develops in ~80% of infected individuals and although
the acute phase of infection is frequently asymptomatic or associated with mild
and non-specific symptoms, these patients are at risk for developing chronic
liver disease (Alter and Seeff, 2000). Approximately 20% of chronic carriers
will develop cirrhosis, and some of these cases will progress to hepatocellular
carcinoma. Consequently, HCV-induced chronic liver disease is now recognized
as the leading indication for orthotopic liver transplantation in the United States
(Fishman et al., 1996).

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Fig. 1. Structure of the HCV genome and Con1-derived bicistronic replicon, location of adaptive
mutations in the HCV polyprotein of subgenomic replicons, and the positions of these mutated
residues in the crystal structures of the NS3 protease, the NS3 helicase, and the NS5B RdRp. (A)
(Top) Schematic of the complete HCV genome. The 5' and 3' NTRs flank the ORF (open box) with
the structural proteins located in the N-terminal portion of the polyprotein and the remainder encoding
the non-structural proteins. (Bottom) Structure of the selectable Con1 bicistronic replicon composed
of the 5' NTR, the first 12 amino acids of the capsid-coding region (open box) fused to the Neo gene

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Hepatitis C viruses have a high level of genetic heterogeneity and thus have been
grouped by their degree of sequence identity into six separate genotypes and
further divided into numerous subtypes (Simmonds et al., 1993; Robertson et al.,
1998). Geographic distribution and responses to current therapy differ between
genotypes. Genotypes 1a and 1b are the most prevalent in the United States and
Western Europe, followed by infections with genotype 2 and 3 strains. The only
licensed therapy for chronic hepatitis C infection is polyethylene glycol (PEG)-
conjugated interferon (IFN)-α given in combination with the guanosine analog
ribavirin; while ~90% of patients persistently infected with HCV genotypes 2 and
3 clear the virus, only 50% of patients infected with HCV genotypes 1, 4, 5, and
6 mount a sustained response (Poynard et al., 2003). Clearly, there is a need for
the development of more effective therapeutic strategies to improve the clinical
treatment of HCV-associated hepatitis.

HCV has been classified as the sole member of the genus Hepacivirus within the
Flaviviridae family, which also includes the classical flaviviruses, such as West
Nile and yellow fever viruses, and the animal pestiviruses, such as bovine viral
diarrhea virus (BVDV). Like these related viruses, HCV is enveloped with a
positive-sense, single-stranded RNA genome. The HCV genome is ~9.6 kb in length
and consists of a 5' non-translated region (NTR) and a long open reading frame
(ORF) encoding all the virus-specific proteins followed by a 3' NTR, comprised
of a short variable sequence, a poly(U)/polypyrimidine [poly(U/UC)] tract, and a
highly conserved terminal sequence (Fig. 1A). Translation of the genomic RNA
is mediated by an internal ribosome entry site (IRES) located within the 5' NTR
(reviewed in Rijnbrand and Lemon, 2000). The resulting polyprotein precursor
of about 3000 amino acids is co- and post-translationally cleaved into at least 10

(Neo; black box), the EMCV IRES (EMCV; solid line), the NS3-5B coding region (open box), and
the 3' NTR structure. (B) Schematic representation of the NS3 to NS5B coding region, sufficient for
autonomous replication of subgenomic replicons in Huh-7 cells. The protease (P) and helicase (H)
domains of NS3 are indicated along with the locations of the ISDR (shaded box) and the adaptive 47
amino acid deletion. The highly adaptive mutations in NS4B, NS5A, and NS5B and the amino acid
substitutions in NS3 that act synergistically with these mutations to enhance subgenomic replication
are shown to the right of the HCV NS3-5B polyprotein. The highly adaptive S2204I substitution in
NS5A is highlighted in bold and those residues in NS3 (E1202G and T1280I) and in NS4B (K1846T)
or NS5A (S2197P) that synergistically enhance replication are shown in italics. (C) Space-filling view
of the NS3/4A serine protease (Protein Database 1A1R; Kim et al., 1996). The adaptive mutations
depicted in panel B, the position of the active-site residues and the location the NS4A cofactor peptide
are indicated. (D) The 3D structure of the NS3 helicase domain complexed with a single-stranded
DNA oligonucleotide (Protein Database 1A1V; Kim et al., 1998). The oligonucleotide, the conserved
DECH motif implicated in coupling NTP hydrolysis to nucleic acid unwinding, and the adaptive
mutations are highlighted. Residue G1304 is partially buried in the molecule and therefore is not
visible in the space-filling model shown. (E) Space-filling model of the NS5B RdRp ectodomain
(Protein Database 1C2P; Lesburg et al., 1999). The putative RNA binding groove is labeled and the
adaptive mutation is highlighted. The color version of this figure can be found at http://blightlab.
wustl.edu/chapter11/.

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Table 1. Summary of the enzymatic functions of the HCV non-structural proteins.

Function
NS2 autoprotease with the N-terminus of NS3 (NS2-3)
NS3 serine protease (N-terminus); NTPase/helicase (C-terminus)
NS4A cofactor for NS3 serine protease
NS4B unknown
NS5A unknown
NS5B RNA-dependent RNA polymerase (RdRp)

different products by a combination of host cell signal peptidases and two viral
proteases. At the N-terminus are the structural proteins C (capsid), E1, and E2
(envelope glycoproteins) followed by the short hydrophobic peptide, p7. The C-
terminal two-thirds of the polyprotein comprise the non-structural proteins (NS)
2, 3, 4A, 4B, 5A, and 5B (Fig. 1A). In addition, the frameshift (F) or alternative
reading frame protein (ARFP), encoded in an overlapping reading frame within
the N-terminus of the HCV polyprotein, is synthesized by ribosomal frameshift
(Walewski et al., 2001; Xu et al., 2001; Varaklioti et al., 2002).

As discussed later, the non-structural proteins NS3-5B are sufficient for subgenomic
replicon replication in cell culture (Lohmann et al., 1999). These proteins are
presumed to function as structural and enzymatic components of the HCV
replication complex or replicase, and the enzymatic properties of the non-structural
proteins are fairly well defined (Table 1 and reviewed in Reed and Rice, 1999;
Blight et al., 2002a). The N-terminus of NS3 is a serine protease that forms a stable
complex with its cofactor NS4A to mediate cleavage of the HCV polyprotein at the
NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions. The C-terminal two-thirds of NS3
harbors an RNA nucleoside triphosphatase (NTPase)/helicase activity capable of
unwinding nucleic acid duplexes. How the NS3 helicase/NTPase contributes to the
RNA replication process is currently unknown, although it is thought to unwind
regions of extensive secondary structure in the template or double-stranded RNAs
resulting from synthesis of the complementary negative-sense RNA intermediate
(see below). NS5B, the C-terminal cleavage product of the polyprotein, is the
RNA-dependent RNA polymerase (RdRp). The roles of NS4B and NS5A in RNA
replication, however, are less clear. NS4B is an integral membrane protein that
induces a distinct membrane alteration, designated the membranous web (Egger
et al., 2002). NS5A is phosphorylated predominantly on serine residues by one or
more unidentified cellular kinases producing two NS5A phosphoprotein variants;
basal- (p56) and hyper- (p58) phosphorylated forms of NS5A (Kaneko et al.,
1994; Tanji et al., 1995; Reed et al., 1997). Surprisingly, NS2 was not required for
subgenomic replication in cell culture (Lohmann et al., 1999) and thus the role of
NS2 in the viral life cycle is not completely clear, although it has been shown to

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form a distinct autoprotease with the serine protease domain of NS3 (NS2-3; Table
1) responsible for cleavage at its own C-terminus (Grakoui et al., 1993; Hijikata
et al., 1993).

The mechanisms of viral attachment and cellular entry and the precise intracellular
steps in HCV RNA replication, virus assembly, and virion release are largely
unknown due to the previous lack of suitable cell culture systems for HCV. Some
details have begun to emerge since the development of subgenomic replicons
that recapitulate the intracellular steps of RNA replication (Fig. 4). Briefly, input
positive-sense HCV RNA is translated and the resultant polyprotein is processed into
the individual HCV proteins. The non-structural proteins assemble in association
with intracellular membranes into the replication complex that transcribes the input
RNA molecule to generate a complementary negative-sense RNA intermediate that
presumably remains base-paired with its template. This double-stranded replicative
form is transcribed asymmetrically, leading to the preferential accumulation of
positive-sense RNAs that are then available for further translation or synthesis of
the negative-sense RNA intermediate.

Since the molecular cloning of the HCV genome 16 years ago, there has been a
great deal of progress in defining HCV genome structure and protein function.
However, the lack of a reliable and robust cell culture system has presented a major
obstacle to studies on the viral life cycle and for developing effective antiviral
drugs. These hurdles have been overcome by the development of subgenomic and
genomic replicons for HCV. In this chapter, we will describe the development of
the HCV replicon system along with the most recent advances and applications
of this system.

DEVELOPMENT OF HCV REPLICONS

Numerous attempts have been made to propagate HCV in cell culture by infection
with virus-containing inoculum. Replication has been detected in hepatoma, B- and
T-cell lines, primary cultures of human or chimpanzee hepatocytes, and peripheral
blood mononuclear cells; however, replication levels were frequently transient
and always so low that HCV RNA synthesis could only be monitored by highly
sensitive reverse transcription (RT)-PCR assays and thus were not amenable to
detailed studies of HCV replication (reviewed in Bartenschlager and Lohmann,
2001). For many positive-sense RNA viruses, including the closely related flavi-
and pestiviruses, productive replication can be efficiently launched by transfecting
permissive cells with genomic RNAs transcribed in vitro from cloned virus
genomes. With this approach, the viral entry and uncoating steps are bypassed;
however, transfection of HCV RNAs transcribed from cDNA clones with proven
infectivity never reproducibly established HCV replication in the many cell lines
tested (reviewed in Bartenschlager and Lohmann, 2001). This was most likely

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due to the generally low levels of replicated RNA and was further complicated
by the difficulty of distinguishing input RNA from plus strands produced by RNA
replication (Fig. 4).

Although HCV is notoriously difficult to grow in cell culture, HCV subgenomic

replicons that efficiently replicate in a human hepatoma cell line, Huh-7, have been
developed (Lohmann et al., 1999). The development of this system was inspired
by the observation that the structural proteins were not required for replication of
several positive-sense RNA viruses including flavi- and pestiviruses, and by the
successful design of self-replicating replicons for the flavivirus Kunjin (Khromykh
and Westaway, 1997) and the pestivirus BVDV (Behrens et al., 1998). The first
generation functional HCV replicons were derived from the consensus Con1 cDNA
that was isolated from the liver of a patient chronically infected with a genotype 1b
strain and comprised: (i) the HCV 5' NTR and the first 12 codons of the capsid protein
fused in-frame with the selectable marker gene, neomycin phosphotransferase (Neo),
which upon expression confers resistance to the cytotoxic drug G418; (ii) the IRES
element from encephalomyocarditis virus (EMCV), which directs translation of the
HCV non-structural proteins; and (iii) the HCV 3' NTR. Fig. 1A depicts the structure
of the bicistronic replicon encoding the HCV polyprotein NS3-5B. Transfection of
Huh-7 cells with transcripts synthesized in vitro and selection with G418 resulted in
a low number of surviving cell colonies. Independent G418-resistant cell colonies
harbored replicons that replicated RNA to high levels (1,000-5,000 copies of positive-
sense HCV RNA per cell), while the negative-sense replicative intermediate was
5-10-fold lower, consistent with asymmetric RNA synthesis. Autonomous HCV
replication was verified by the efficient labeling of HCV RNA with [3H] uridine
in the presence of actinomycin D, an inhibitor of DNA- but not RNA- dependent
RNA polymerases (Lohmann et al., 1999). HCV non-structural proteins in G418-
selected cell clones localized exclusively to the cytoplasm in close association with
membranes of the endoplasmic reticulum (ER) (Pietschmann et al., 2001; Mottola
et al., 2002), a predicted site of HCV RNA replication (discussed below).

IDENTIFICATION OF ADAPTIVE MUTATIONS

Despite the high levels of subgenomic RNA replication within a selected cell clone,
G418 resistance arose in a very low frequency of transfected cells (~1 colony per
106 transfected cells; Lohmann et al., 1999; Blight et al., 2000). This was due to two
restrictions: first, replicon RNAs had to acquire adaptive mutations to efficiently
replicate in the Huh-7 cell line; and second, only a low number of cells in the culture
support efficient HCV replication (discussed in detail below). Sequence analysis of
Con1-derived HCV RNAs replicating in cell clones after G418 selection identified
in most cases at least one mutation in the non-structural coding region, but not in the
highly conserved 5' or 3' NTRs (Blight et al., 2000; Krieger et al., 2001; Lohmann
et al., 2001; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). The

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impact of individual mutations on HCV RNA replication was tested by engineering

mutations into the parental replicon and determining the number of G418-resistant
colonies after transfection of a defined amount of in vitro transcribed RNA, or by
transient replication assays. As discussed below, most mutations were found to
enhance RNA replication in Huh-7 cells; however, the degree of adaptation was
highly dependent on the particular substitution.

Highly adaptive mutations lie within the NS4B, NS5A, and NS5B coding regions,
with the majority clustering in NS5A, just upstream of the sequence termed the IFN
sensitivity-determining region (ISDR; Fig. 1B), a region that has been implicated
in the effectiveness of IFN treatment (Enomoto et al., 1995; Enomoto et al., 1996).
Highly adaptive amino acid substitutions have been identified at nine positions in
Con1 NS5A (Fig. 1B; Blight et al., 2000; Krieger et al., 2001; Guo et al., 2001;
Lohmann et al., 2003; Lanford et al., 2003). The most efficient adapted replicon
contains a single serine to isoleucine substitution at position 2204 in NS5A (S2204I;
Fig. 1B) and establishes replication in ~10% of transfected Huh-7 cells (Blight et
al., 2000). The >10,000-fold improvement in colony-forming efficiency compared
to the parental Con1 replicon was sufficient for the detection of HCV RNA and
proteins shortly after RNA transfection. By 96 hours, HCV RNA levels were almost
500-fold higher than a replicon carrying a lethal mutation in the NS5B RdRp (Blight
et al., 2000). In addition to amino acid substitutions, an in-frame deletion of 47
amino acids encompassing the putative ISDR (∆2207-2254; Fig. 1B) (Blight et al.,
2000) and a deletion of the serine residue at position 2201 (∆S2201; Fig. 1B) (Guo
et al., 2001) also enhance replicon replication in Huh-7 cells.

Based on the predicted topology of the integral membrane protein NS4B, amino
acid substitutions reside in distinct cytoplasmic domains of this protein (Guo et al.,
2001; Lohmann et al., 2003). Mutations at these sites have a strong impact on Con1
replication with a K1846T substitution enhancing replication to a greater extent
than V1897A (Lohmann et al., 2003). In contrast to the highly adaptive mutations
in NS4B and NS5A, amino acid substitutions within NS5B are only moderately
enhancing. For instance, a single amino acid substitution at position 2884 (R2884G;
Fig. 1B and E) increases G418-colony formation by ~500-fold compared to the
parental sequence (Lohmann et al., 2001).

Another cluster of adaptive mutations (Fig. 1B) mapped to the solvent-accessible

surface of the NS3 crystal structure and at a distance from the active sites of the
protease and helicase (Fig. 1C and D). Interestingly, the mutations lying within
NS3 were always found in conjunction with highly adaptive substitutions (Blight
et al., 2000; Krieger et al., 2001; Lohmann et al., 2001; Guo et al., 2001; Lohmann
et al., 2003; Lanford et al., 2003). By themselves, these NS3 mutations have
minimal or no impact on replicon replication (Krieger et al., 2001; Lohmann et

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al., 2001; Lohmann et al., 2003; Lanford et al., 2003), but can enhance replication
synergistically when combined with each other or with highly adaptive mutations
in NS4B, NS5A, or NS5B (Krieger et al., 2001; Lohmann et al., 2001; Lohmann et
al., 2003; Lanford et al., 2003). To illustrate this point, two amino acid substitutions
in NS3 (E1202G and T1280I) together with either K1846T in NS4B (Lohmann et
al., 2003) or S2197P in NS5A (Krieger et al., 2001) enhance Con1 replication to
levels above those observed for the replicons harboring the single NS4B or NS5A
adaptive mutations. In contrast, combinations of highly adaptive mutations in NS4B,
NS5A, and NS5B are antagonistic, albeit to different extents. Combining highly
adaptive mutations in NS5A with each other (Blight et al., 2002b; Lohmann et
al., 2003) or with the NS5B R2884G substitution (Lohmann et al., 2003) severely
impair or completely abolish replication. Thus, it appears that the mechanism(s)
of cell culture adaptation achieved by mutations in NS3 is different from the one
exerted by substitutions in NS4B, NS5A, and NS5B.

At this stage, we can only speculate about the mechanism(s) for adaptive mutation-
enhanced replication and the synergy between mutations in NS3 and those in NS4B,
NS5A, or NS5B. Most mutations conferring cell culture adaptation have not been
found in natural isolates of HCV and almost invariably target amino acid residues
that are conserved between different HCV genotypes, suggesting that mutations
represent a specific adaptation to the Huh-7 cell environment. Given that adaptive
mutations reside on the surface of the available crystal structures for NS3 and
NS5B (Fig. 1C-E) and do not affect the active sites of these enzymes, it is assumed
that mutations modulate interactions among viral proteins and/or between viral
and cellular components of the HCV replication complex. There is accumulating
evidence that the suppression of NS5A hyperphosphorylation may represent a
mechanism of replicon adaptation. In support of this, Con1 subgenomic RNA no
longer requires adaptive mutations to efficiently replicate when Huh-7 cells are
treated with inhibitors that block NS5A hyperphosphorylation (Neddermann et al.,
2004). Additionally, site-directed mutagenesis of the serine residues involved in
NS5A hyperphosphorylation led to a decrease in p58 formation with a corresponding
increase in HCV replication (Appel et al., 2005b). Similarly, highly adaptive
mutations targeting serine residues in NS5A either ablate (eg. S2204I; Blight et al.,
2000) or impair NS5A hyperphosphorylation (eg. S2197P/C; Blight et al., 2000).
In addition, the replication-enhancing mutations in NS4B also reduce the level
of NS5A hyperphosphorylation (Evans et al., 2004b; Appel et al., 2005b). Thus,
impaired NS5A hyperphosphorylation is a critical requirement for efficient Con1
RNA replication in Huh-7 cells. Perhaps hyperphosphorylation of NS5A performs
a regulatory role in the HCV life cycle and adaptive mutations that suppress NS5A
hyperphosphorylation prevent the dissociation of the replication complex, thereby
allowing the establishment of ongoing efficient replication (Evans et al., 2004b;
Appel et al., 2005b). Alternatively, it has been shown that antiviral pressures of the

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host cell triggered by HCV RNA replication contribute to the acquisition of adaptive
mutations. Specifically, when a Huh-7 cell clone supporting replication of an IFN-
sensitive Con1 subgenomic RNA was maintained long term in culture, mutations
accumulated throughout the HCV-coding region. The fitness of this replicon variant
was significantly enhanced and was resistant to the host defenses triggered by
productive replication and by IFN-α treatment (Sumpter Jr. et al., 2004).

As observed for many other viruses, especially those with high mutation rates,
passages in cell culture for prolonged periods of time can result in the accumulation
of mutations that often improve virus replication in vitro but frequently lead to
attenuation in vivo. Similarly, cell culture-adaptive mutations that facilitate efficient
HCV replication in Huh-7 cells give rise to highly attenuated phenotypes in vivo.
Intrahepatic inoculation of chimpanzees, the only recognized animal model for
HCV infection, with full-length Con1 genomes harboring three cell culture-adaptive
mutations (E1202G and T1280I in NS3 and S2197P in NS5A; Fig. 1B) failed to
develop a productive infection (Bukh et al., 2002). A Con1 genome with the single
S2197P substitution in NS5A replicated poorly, and one week after inoculation
circulating HCV genomes were detectable but had reverted to the original wild-type
Con1 sequence (Bukh et al., 2002). Thus, the attenuation in vivo of Con1 genomes
carrying cell culture-acquired mutations may explain why Huh-7 cells supporting
full-length Con1 replication do not produce infectious virus particles (Pietschmann
et al., 2002; Blight et al., 2002b and discussed below).

GENERATION OF REPLICONS FROM OTHER HCV ISOLATES

Slight variations in HCV sequence can dramatically alter the replicative ability of
engineered replicons and as a result creating functional subgenomes for different
HCV isolates has not been straightforward. Of the six HCV genotypes, viable
replicons have only been reported for genotype 1 and 2 strains. Six genotype 1b
isolates - Con1 (discussed above), HCV-N (Guo et al., 2001; Ikeda et al., 2002),
HCV-BK (Grobler et al., 2003), HC-J4 (Maekawa et al., 2004), 1B-2/HCV-O (Kato
et al., 2003a), and 1B-1/M1LE (Kishine et al., 2002) - productively replicate in
Huh-7 cells. Replication-competent genotype 1a replicons are derived from the
Hutchinson strain (H77 or HCV-H; Blight et al., 2003; Gu et al., 2003; Grobler
et al., 2003; Yi and Lemon, 2004; Liang et al., 2005) and efficient replication of
JFH-1, classified as a genotype 2a virus, has been also demonstrated in cell culture
(Kato et al., 2003b).

GENOTYPE 1b
For many years, the only HCV replicons able to autonomously replicate in cultured
Huh-7 cells were derived from the Con1 strain. This restriction has now been
overcome by the development of replication-competent subgenomic RNAs derived
from independent genotype 1b isolates. Unlike the Con1 replicons, cell culture

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adaptation does not appear to be required for efficient replication of subgenomes

derived from the HCV-N isolate, nor for the G418 selection of Huh-7 clones
(Guo et al., 2001; Ikeda et al., 2002). Instead, a unique four amino acid insertion
naturally present within the ISDR of the HCV-N NS5A protein was sufficient for
persistent replication in Huh-7 cells. Removal of these four amino acids from the
HCV-N replicon drastically reduced its capacity to confer resistance to G418, but
replication could be restored by incorporation of the highly adaptive Con1 mutation,
S2204I in NS5A (Ikeda et al., 2002). Thus, this natural four amino acid insertion
in HCV-N NS5A behaves like a cell culture-acquired adaptive mutation. For the
HCV-BK strain, systematic mutagenesis of the NS3 coding region showed that
efficient replicon replication required a mutation in the helicase domain of NS3
(R1496M) in addition to the S2204I substitution in NS5A (Grobler et al., 2003).
NS5A adaptive mutations S2197P, S2204I, or ∆S2201 (Fig. 1B) were necessary
for productive replication of chimeric HC-J4 replicons containing the 5' NTR and
first 75 amino acids of NS3 from the Con1 strain (Maekawa et al., 2004). While
the above genotype 1b replicons were derived from cDNA clones, subgenomic
replicons have also been constructed from HCV genome RNA replicating at low
levels in cultured cells infected with human serum containing genotype 1b HCV.
A very low number of G418-resistant colonies was obtained after transfection of
Huh-7 cells, or more permissive Huh-7 sublines, with subgenomic replicons derived
from the human T-cell line MT-2C infected with HCV isolate M1LE (Kishine et al.,
2002) or from human non-neoplastic hepatocytes (PH5CH8) infected with HCV
isolate HCV-O (Kato et al., 2003a). Subgenomes replicating in G418-selected
cell clones harbored mutations in NS3 as well as NS4B or NS5A. Although the
adaptive advantage of these mutations for M1LE and HCV-O replication has not
been determined, substitutions at these positions in the Con1 polyprotein have been
shown to enhance Con1 subgenomic replication (Lohmann et al., 2001; Lohmann
et al., 2003; Lanford et al., 2003).

GENOTYPE 1a
The identification of efficiently replicating replicons corresponding to genotype 1a
strains has proven even more challenging than generating functional genotype 1b
subgenomic RNAs. Attempts to construct a replication-competent replicon from the
HCV-1 infectious clone have been unsuccessful, despite the inclusion of adaptive
mutations identified in the genotype 1b Con1 replicon (Lanford et al., 2003). Similar
negative results were obtained for the H77 strain until highly permissive cell lines
were isolated (Blight et al., 2003; Grobler et al., 2003) or H77-Con1 chimeric
replicons were constructed (Gu et al., 2003; Yi and Lemon, 2004). Although
intrahepatic inoculation of H77 RNA is associated with high viremia during the
acute phase of infection in the chimpanzee (Kolykhalov et al., 1997; Yanagi et al.,
1997), replicons derived from this infectious H77 molecular clone require at least
two adaptive mutations to productively replicate in cell culture. Interestingly, the

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single adaptive mutation S2204I in NS5A that was identified in the Con1 replicon
was a crucial prerequisite for obtaining G418-resistant colonies supporting H77
replication (Blight et al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon,
2004), indicating that at least one of the Con1 adaptive mutations is also effective
in a genotype 1a sequence.

Transfection of a highly permissive Huh-7 subline, Huh-7.5 (Blight et al., 2002b

and see below), with S2204I-containing H77 replicons allowed the establishment
of the first G418-resistant colonies supporting H77 replication (Blight et al., 2003).
Analysis of H77 RNAs replicating in these G418-selected cell clones identified a
second amino acid substitution in the helicase domain of NS3 (A1226D or P1496L;
Fig. 2A and C). Both of these NS3 mutations, when combined individually with
S2204I in NS5A, increased the replicative capacities of subgenomic H77 RNA with
the greatest enhancement seen with the P1496L substitution. Similarly, Grobler et al.
(Grobler et al., 2003) independently found that P1496L (or S1222T) together with
S2204I is sufficient for productive replication of H77 RNA in a hyper-permissive
Huh-7 subline, MR2. Like the synergistic NS3 mutations identified in the Con1
strain, H77 residues S1222, A1226, and P1496 map to the surface of the NS3
helicase and are not located in the nucleotide, metal, or nucleic acid binding sites
(Fig. 2C). Additionally, replacement of proline with leucine at position 1496 has
no effect on the in vitro unwinding activity of purified NS3 helicase (Grobler et al.,
2003), suggesting that these mutations are involved in mediating interactions with
viral or cellular factors rather than increasing the enzymatic capacity.

Alternatively, the requirements for productive H77 replication in the parental Huh-7
cell line were defined through the construction of chimeric replicons between Con1
and H77 sequences harboring S2204I in NS5A (Gu et al., 2003; Yi and Lemon,
2004). One study (Yi and Lemon, 2004) identified adaptive mutations within NS3,
NS4A, and NS5A that act cooperatively to enhance H77 replication. Maximal non-
chimeric H77 replication was achieved with a combination of mutations Q1067R
and V1655I in NS3, K1691R in NS4A, and K2040R and S2204I in NS5A (Fig. 2A).
In contrast to the NS3 mutations found in the helicase domain (Fig. 2C; Blight et al.,
2003; Grobler et al., 2003), the substitutions identified in NS3 by Yi and Lemon (Yi
and Lemon, 2004; Fig. 2A) are located in close proximity to the protease active site
in the NS3/4A crystal structure (Q1067 and G1188; Fig. 2B) or in the P3 position of
the NS3/4A cleavage site potentially influencing substrate recognition during cis-
cleavage at this junction (V1655; Fig. 2A). Furthermore, K1691 in NS4A is located
immediately downstream of the sequence involved in complex formation with NS3.
Thus, these mutations (Yi and Lemon, 2004) may facilitate HCV replication via a
mechanism different from those amino acid substitutions previously identified in
the helicase domain of NS3 (Blight et al., 2003; Grobler et al., 2003). Similarly,
another laboratory (Gu et al., 2003) achieved efficient replication of a replicon that

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Blight and Norgard

Fig. 2. Location of adaptive mutations that enhance H77 replicon replication in Huh-7 cells. (A) The
NS3 to NS5B polyprotein with the positions of individual mutations proven to facilitate efficient
replication in combination with S2204I in NS5A (bold) shown on the right. The protease (P) and
helicase (H) domains in NS3 and the ISDR in NS5A are illustrated. Solvent-accessible surface of
the NS3/4A protease (B) and NS3 helicase (C) crystal structures. The adaptive mutations and other
significant features are highlighted as described in Fig. 1. The coordinates of these structures were
retrieved from protein database under accession number 1A1R (Kim et al., 1996) and 1A1V (Kim
et al., 1998) for the models shown in B and C, respectively. This figure can be viewed in color at
http://blightlab.wustl.edu/chapter11/.

was predominantly H77 derived, except the 5' NTR and N-terminal 75 amino acids
of NS3 were from the Con1 genotype 1b strain. In the single G418-resistant cell
clone analyzed, four amino acid changes were identified across NS3, NS5A, and
NS5B; however, it is unclear which mutations or combination of mutations was
responsible for augmenting chimeric or non-hybrid replication (Gu et al., 2003).

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 HCV Replicons

In complete contrast, an H77-derived replicon encoding the puromycin N-

acetyltransferase (PAC) gene instead of the Neo cassette (Fig. 3A) does not require
adaptive mutations to autonomously replicate and confer resistance to puromycin
in Huh-7 cells, although S2204I in NS5A did evolve in a minority of cell clones
(Liang et al., 2005). Additionally, unlike the studies discussed above (Blight et
al., 2003; Gu et al., 2003; Grobler et al., 2003; Yi and Lemon, 2004), inclusion of
S2204I in NS5A did not significantly improve the colony-forming efficiency of
PAC-expressing replicons (~3-fold improvement; Liang et al., 2005). The ability
of PAC-expressing H77 replicons to establish replication in Huh-7 cells without
adaptive mutations is puzzling and may reflect the different selective pressure.
Perhaps cellular environments that are conducive to non-adapted H77 replication are
more efficiently selected by puromycin than G418. In support of this, transfection
of total cellular RNA extracted from a replicon-containing cell clone led to a 50-
fold increase in puromycin-resistant colonies, suggesting that translation of cellular
mRNAs initially introduced with replicon RNA created an intracellular milieu in
naïve Huh-7 cells favorable for the establishment of HCV replication.

GENOTYPE 2a
Subgenomic replicons derived from the genotype 2a JFH-1 clone, initially isolated
from a patient with fulminant hepatitis, represent the only non-genotype 1 sequence
currently capable of efficient replication in cell culture (Kato et al., 2003b).
Interestingly, JFH-1 subgenomes replicate with high efficiency in Huh-7 cells in
the absence of adaptive mutations; the G418-resistant colony-forming ability of
the unmodified bicistronic replicon is 60-fold higher than a Con1 subgenomic
RNA harboring highly adaptive mutations (Kato et al., 2003b). Although adaptive
mutations are not a prerequisite for efficient JFH-1 replication, amino acid changes
in the replicase proteins were identified in the majority of G418-selected replicon-
containing Huh-7 clones. Of those tested, one mutation, H2476L in NS5B, enhanced
the G418 transduction efficiency by only 3-fold, which is well below the level
of enhancement seen for single highly adaptive Con1 mutations (Blight et al.,
2000; Guo et al., 2001; Lohmann et al., 2003; Lanford et al., 2003). Nonetheless,
the colonies derived from JFH-1 replicons containing this NS5B mutation were
significantly larger than those obtained after transfection of unmodified JFH-1
subgenomes (Kato et al., 2003b), suggesting that this mutation confers a higher
replication phenotype in Huh-7 cells. Furthermore, the high replication efficiency
of unmodified JFH-1 replicons allowed HCV RNA and proteins to be monitored
in transient replication assays (Fig. 4). Based on the data presented by Kato and
coworkers (Kato et al., 2003b), the JFH-1 subgenomic RNA is the most efficient
replicon tested so far. Additionally, it appears that adaptive mutations may not always
be necessary for efficient replication in cell culture. Instead, the requirement for
adaptive mutations is dependent on the individual HCV isolate. Identification of the
JFH-1 determinants that promote this high level of RNA replication could provide

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Fig. 3. Organization of replication-competent HCV replicons reported so far. (A) Antibiotic

selectable replicons. (Top) Biscistonic HCV replicons are composed of the 5' NTR, a small portion
of the capsid-coding region (open box), an antibiotic resistance gene (neomycin phosphotransferase
[Neo], blasticidin S deaminase [blast], hygromycin phosphotransferase [hygro], or puromycin
acetyltransferase [PAC]; black box), the EMCV IRES (EMCV; solid line), the NS3-5B coding region
and the 3' NTR. (Bottom) Monocistronic hygromycin selectable replicon consisting of the 5' NTR, the
first 16 amino acids of the capsid fused to the hygromycin phosphotransferase gene (hygro; black box),
ubiquitin coding sequence (Ub), the HCV non-structural proteins NS3 to NS5B (open box), and the
3' NTR. (B) Replicons encoding reporter genes or a transactivator of SEAP expression. The 5' and 3'

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insights into the mechanisms of HCV RNA replication. Replication of a genotype

that diverges from genotype 1 isolates by ~30% not only represents an important
advance for replication studies, but also for drug discovery efforts.

DEVELOPMENT OF ALTERNATIVE REPLICON DERIVATIVES

The identification of adaptive mutations that dramatically enhance genotype 1 HCV
replication in cell culture has made it possible to explore alternative drug resistance
genes (Fig. 3A; Frese et al., 2002; Evans et al., 2004a; Liang et al., 2005; Appel et
al., 2005a) and develop transient replication assays utilizing replicon derivatives
expressing or activating the expression of easily quantifiable reporter enzymes
(Fig. 3B; Krieger et al., 2001; Yi et al., 2002; Lohmann et al., 2003; Murray et al.,
2003; Ikeda et al., 2005). Furthermore, robustly replicating monocistronic replicons,
containing one translation module and eliminating non-HCV sequences (Fig. 3C;
Blight et al., 2003), have also been generated, as well as replication-competent
full-length RNAs that stably or transiently replicate in Huh-7 cells (Fig. 3D; Ikeda
et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003; Yi
and Lemon, 2004; Ikeda et al., 2005).

DRUG RESISTANCE GENES

Engineering alternative drug resistance genes may permit faster selection of
replication-positive cells as well as allow different replicon sequences to be
sequentially or simultaneously selected within the same cell. Neo-encoding Con1
replicons have been successfully selected in the same cell with Con1 bicistronic
replicons encoding either blasticidin S deaminase (Evans et al., 2004a) or
hygromycin phosphotransferase (Appel et al., 2005a) genes in place of the Neo
cassette (Fig. 3A). This approach found: (i) no evidence of recombination between
different subgenomes (Evans et al., 2004a; Appel et al., 2005a); (ii) a high level
of competition between subgenomic RNAs, suggesting that host cell machinery
required for HCV replication is limiting (Evans et al., 2004a) and; (iii) replicons
harboring lethal mutations in NS3, NS4B, or NS5B or mutations that disrupt the
structure of the N-terminal membrane anchor of NS5A could not be rescued by
co-expression of functional subgenomic RNAs (Evans et al., 2004a; Appel et al.,
2005a). Thus, it appears that HCV replication complexes are relatively closed

NTR structures are shown, the first 12 amino acids of capsid and the HCV ORF are depicted by open
boxes and the reporter genes, firefly luciferase (fLUC), β-lactamase (bla), GFP, and renilla luciferase
(rLUC) are represented by hatched boxes. The Neo gene (Neo; black box), the EMCV IRES (EMCV;
solid line), the poliovirus IRES (Polio; dashed line), ubiquitin (Ub; shaded box), the HIV tat protein
(tat; hatched box), and the foot and mouth disease virus protease 2A (2A; shaded box) are depicted.
The curved arrow indicates the site of autocatalytic 2A-mediated cleavage. (C) Monocistronic HCV
subgenome containing the 5' NTR, the capsid (C)-coding region fused to the NS2-5B ORF (open
box) followed by the 3' NTR. (D) Full-length HCV replicons encoding the entire HCV polyprotein
coding sequence (C-NS5B; open box). The 5' and 3' NTRs, the Neo gene (Neo; black box), the renilla
luciferase gene (rLUC; hatched box) and EMCV IRES (EMCV; solid line) are illustrated.

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Blight and Norgard

structures preventing or limiting the exchange of viral proteins. However, deletions

in NS5A or multiple mutations affecting potential phosphorylation sites in the
central region of NS5A could be complemented in trans, suggesting that NS5A is
loosely associated with the intracellular membranes that provide the scaffold of
the HCV replication complex (Appel et al., 2005a). Alternatively, a monocistronic
replicon encoding the hygromycin phosphotransferase gene establishes productive
replication in Huh-7 cells and confers resistance to hygromycin (Frese et al.,
2002). In this replicon, ubiquitin was inserted in-frame between hygromycin
phosphotransferase and the NS3-5B coding region so that translation of the entire
ORF is directed by the HCV 5' NTR and NS3 is released from the polyprotein by
a cellular ubiquitin carboxyl-terminal hydrolase-mediated cleavage event at the
ubiquitin/NS3 junction (Fig. 3A).

REPORTER GENES
Assays for colony formation are time consuming and assume that the frequency of
drug-resistant colonies observed with a given replicon is directly proportional to
its intrinsic replication activity. With the identification of adaptive mutations that
facilitate efficient HCV replication, transient RNA replication assays that allow
a more rapid and direct analysis of relative replication efficiencies have been
developed. Reporters such as luciferase and ß-lactamase, as well as a transactivator
inducing secreted alkaline phosphatase (SEAP), have been used to monitor
replication at early times after transfection of Huh-7 cells.

Firefly luciferase has successfully replaced the Neo gene in Con1 (Krieger et al.,
2001) and HCV-O (Ikeda et al., 2005) bicistronic replicons (Fig. 3B), thus enabling
replication to be monitored at various times following transfection by measuring the
luciferase activity relative to a polymerase-defective replicon. After 48-72 hours, the
luciferase activities seen with an adapted Con1 replicon are about 100-fold higher
than the negative control (Krieger et al., 2001). The luciferase activity directly
correlates with the levels of HCV RNA synthesis, demonstrating that luciferase
is a reliable marker of replication (Krieger et al., 2001). Enhanced replication
levels, and thus higher luciferase activities (~5-fold), as well as more reproducible
results were achieved by placing luciferase under the translational control of the
IRES from poliovirus instead of the HCV IRES (Fig. 3B; Lohmann et al., 2003).
Although luciferase activity allows the rapid determination of relative replication
levels in the population of transfected cells, it does not provide a direct measure of
the number of cells supporting replication. This restriction has been overcome by
the development of bicistronic Con1 replicons containing a ß-lactamase reporter
(Fig. 3B; Murray et al., 2003). In this strategy, Huh-7 cells supporting active HCV
replication are identified using a cell-permeable fluorescent substrate that is cleaved
by ß-lactamase expressed in the cell, leading to blue fluorescence. More recently, it
has been reported that the C-terminal domain of NS5A (between residues 2370 and

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2412) is dispensable for replicon function (Appel et al., 2005b) and heterologous
sequences can be inserted within this region with only a moderate reduction in
replication (Moradpour et al., 2004b; Appel et al., 2005b). Thus, viable Con1
replicons carrying an in-frame insertion of enhanced green fluorescent protein
(GFP) at position 2356 or 2390 in NS5A were created (Fig. 3B); however, the G418
transduction efficiency of GFP-expressing replicons was ≥25-fold lower than the
parental replicon constructs (Moradpour et al., 2004b). Although the GFP signal is
readily visualized in G418-selected cell clones by fluorescence microscopy allowing
active replication complexes to be tracked in real time (Moradpour et al., 2004b and
see below), it is not clear if the replication competence of these GFP-expressing
replicons is sufficient for the detection and quantification of GFP-positive cells in
transient replication assays. In an independent report a firefly luciferase-expressing
Con1 subgenomic RNA (Fig. 3B) carrying a GFP insertion between positions
2370 and 2412 of NS5A replicated to levels about 100-fold below the parental
replicon that lacked GFP and this lower replication efficiency prevented the direct
visualization of the NS5A-GFP fusion protein at 72 hr post-transfection (Appel
et al., 2005b).

The first cistron of the bicistronic Con1, HCV-N, (Yi et al., 2002) and H77 (Yi and
Lemon, 2004) replicons have been modified to include the human immunodeficiency
virus (HIV) tat protein, a potent transcriptional transactivator of the HIV long
terminal repeat (LTR) promoter (Yi et al., 2002). Briefly, the tat-coding sequence
was fused to a picornaviral 2A protease sequence followed by the Neo selectable
marker (Fig. 3B), such that upon translation, the autocatalytic protease activity of
2A mediates cleavage at its C-terminus, liberating Neo. Replication in transfected
cells leads to the intracellular accumulation of tat, which in turn activates the LTR-
SEAP cassette, which is stably integrated into the genome of the transfected Huh-7
cell. SEAP is subsequently expressed and secreted from replication-positive cells
and five days after transfection of adapted replicons, extracellular SEAP can reach
levels 100-fold above the amount secreted from cells transfected with replication-
defective mutants (Yi et al., 2002).

In addition to the use of these reporter gene replicon systems for rapid determination
of replicative ability, these systems are amenable to high throughput tests including
screening large compound libraries for anti-HCV activity. For instance, the ß-
lactamase replicon system has been successfully adapted to a high-throughput
screening assay to identify inhibitors of HCV replication (Zuck et al., 2004).
Furthermore, replicons carrying firefly or renilla luciferase fused to the neomycin
phosphotransferase gene (Fig. 3B) have been used to assess the effect of human
IFN-α and ribavirin on HCV replication (Tanabe et al., 2004; Ikeda et al., 2005).
Another group determined the level of biologically active IFN-α in sera taken
from HCV carriers undergoing IFN treatment by using cells harboring a bicistronic

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replicon where the first cistron comprises a firefly luciferase-ubiquitin-Neo cassette

(Fig. 3B; Vrolijk et al., 2003).

MONOCISTRONIC SUBGENOMIC REPLICONS

The ability to monitor HCV replication without selection eliminates the requirement
for bicistronic replicons. In fact, heterologous sequences such as the Neo gene
and the EMCV IRES reduce the replicative capacity of Con1- and H77-derived
subgenomic RNAs (Blight et al., 2002b; Blight et al., 2003). Robust replication is
observed with a monocistronic replicon composed of the 5' NTR followed by the
entire capsid sequence fused to the NS2-NS5B coding region, such that cleavage
between capsid and NS2 is mediated by the host cell signal peptidase and translation
is under the control of the HCV IRES (Fig. 3C; Blight et al., 2003). The replication-
competence of this subgenomic RNA, as well as the monocistronic hygromycin
phosphotransferase-encoding replicon described above (Fig. 3A; Frese et al., 2002),
demonstrates that the EMCV IRES is not a requirement for efficient expression
of the HCV replicase region and subgenomic replication in Huh-7 cells. Since
this monocistronic replicon lacks antibiotic resistance or reporter genes (Fig. 3C),
replication in transfected cells can only be monitored by the detection of HCV-
specific RNA and proteins. Quantitative real-time RT-PCR (Blight et al., 2000;
Blight et al., 2002b; Blight et al., 2003) and Northern blot hybridization (Krieger
et al., 2001; Pietschmann et al., 2002; Lohmann et al., 2003; Kato et al., 2003b; Yi
and Lemon, 2004) have been used to analyze HCV RNA synthesis (Fig. 4), and
HCV protein expression has been successfully detected by a collection of assays
including Western blot, fluorescent activated cell sorting (FACS), metabolic labeling
and immunoprecipitation with HCV-specific antisera, and immunostaining of cell
monolayers (Fig. 4; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al., 2003;
Kato et al., 2003b). HCV subgenomic replication can now be studied in the absence
of heterologous sequences, alleviating concerns that the experimental phenotype is
related to the expressed heterologous gene or foreign IRES element.

FULL-LENGTH REPLICONS
Full-length or genomic HCV replicons derived from Con1, H77, HCV-O, and HCV-
N productively replicate in Huh-7 cells or in highly permissive Huh-7 sublines
(Ikeda et al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Blight et al.,
2003; Yi and Lemon, 2004; Ikeda et al., 2005). Full-length HCV RNAs carry the
complete HCV open reading frame (C-NS5B) and replication is dependent on
cell culture-adaptive mutations. Bicistronic derivatives encoding Neo (Ikeda et
al., 2002; Pietschmann et al., 2002; Blight et al., 2002b; Ikeda et al., 2005) or a
renilla luciferase-Neo fusion (Ikeda et al., 2005) have been generated (Fig. 3D),
facilitating the selection of stable G418-resistant cell lines supporting full-length
HCV replication. The number of Huh-7 cells able to support full-length replication
is much lower than that seen for the subgenomic derivative carrying the same
adaptive mutations and the average level of full-length RNA replication is about

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 HCV Replicons

Fig. 4. The intracellular steps involved in HCV RNA replication and the various methods used to
monitor these steps in stable antibiotic-resistant cell clones or in transient replication assays. After
transfection of Huh-7 cells with in vitro transcribed RNAs (lightening bolt), measurable reporters,
transactivators inducing reporter gene expression, antibiotic resistance markers, and the HCV
polyprotein are expressed via IRES-dependent translation (A). (B) The HCV polyprotein is processed
into the individual proteins and the NS3-5B proteins associate to form a membrane-associated
replication complex. (C) Positive-sense HCV RNA is transcribed into a complementary negative-sense
intermediate and progeny positive-sense HCV RNAs then serve as templates for additional negative-
sense RNA synthesis (small open arrows) or further translation (large open arrow). Upon establishment
of HCV replication, foreign genes are expressed to levels allowing antibiotic selection and/or
quantification of reporter gene expression (A). HCV protein production (B) and RNA synthesis (total,
positive- or negative-sense HCV RNA; C) can be measured by the methods shown. Abbreviations:
IP – immunoprecipitation; actD – actinomycin D; FACS – fluorescent activated cell sorting.

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Blight and Norgard

5-fold lower than subgenomic replication (Pietschmann et al., 2002; Blight et al.,
2002b; Blight et al., 2003). So far, there is no evidence of HCV particle assembly
and release from Huh-7 cells supporting replication of Con1 (Pietschmann et al.,
2002; Blight et al., 2002b) or H77 (Blight et al., 2003) full-length RNAs containing
cell culture-adaptive mutations. Although unmodified full-length HCV RNAs
generated from these HCV strains produce infectious virus in the chimpanzee model
(Kolykhalov et al., 1997; Yanagi et al., 1997; Bukh et al., 2002), Con1 genomes
harboring cell culture-adaptive mutations are severely attenuated in vivo (Bukh et
al., 2002), suggesting that adaptive mutations inhibit virus particle assembly. In
support of this, Huh-7 cells supporting replication of a full-length RNA containing
the non-structural proteins from the genotype 2a JFH-1 strain that lacks adaptive
mutations assemble and release infectious virus particles (Bartenschlager et al.,
2004; Heller et al., 2005). Additionally, virus production has also been observed in
Huh-7 cells transfected with plasmid DNA carrying an unmodified infectious full-
length genotype 1b HCV genome flanked by self-cleaving hammerhead ribozymes
to generate the exact 5' and 3' ends of intracellular transcribed RNA (Heller et al.,
2005). The development of cell culture systems supporting the complete virus life
cycle now allows studies directed towards defining the mechanisms of viral particle
assembly (see Chapter 16).

CELL LINES PERMISSIVE FOR HCV REPLICATION

In vivo, hepatocytes are believed to be the major site of HCV replication; however,
some evidence suggests that extrahepatic cells, including lymphocytes, monocytes,
and dendritic cells, may also harbor the virus (Blight and Gowans, 1995; Laskus et
al., 2000; Goutagny et al., 2003). Productive replication of HCV replicons in vitro
appears to be extremely cell-type specific, with human hepatoma Huh-7 cells being
the most permissive cell line identified so far, although, as described below, the
environment within these cells affects the efficiency of HCV replication. Extensive
effort has been devoted to identifying other permissive cell lines and recently, the
cell repertoire was expanded to include additional continuous human hepatoma
cell lines and a murine hepatoma cell line, as well as non-hepatic cell lines such as
the human cervical cancer-derived HeLa cell line and 293 cells established from
human embryonic kidney.

Huh-7 CELL LINE

Although cell culture-adaptive mutations are essential for genotype 1 replication
in Huh-7 cells, there is convincing evidence that the environment within the Huh-7
cell also governs the ability of HCV RNAs to replicate. First, the relative replication
efficiencies of subgenomic RNAs in transient replication assays can vary by as
much as 100-fold between different passages of Huh-7 cells. These differences
are independent of the adaptive mutation(s) introduced into the replicon and are
not due to differences in HCV RNA translation or stability (Lohmann et al., 2003),

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 HCV Replicons

suggesting that efficient replication depends on host cell conditions or specific

cellular factors. Secondly, replication of subgenomic RNAs, despite the inclusion
of highly adaptive mutations, can only be detected in a subset of transfected Huh-7
cells (~10% for the highly adaptive mutation S2204I in NS5A; Blight et al., 2000;
Blight et al., 2002b), suggesting that many Huh-7 cells do not provide an optimal
environment for HCV replication. Additionally, the number of G418-resistant
colonies obtained after transfection of selectable replicons was significantly higher
in cell clones from which the replicon had been eliminated by extended treatment
with IFN-α ("curing") than that observed for naïve Huh-7 cells (Blight et al., 2002b;
Murray et al., 2003; Ikeda et al., 2005). Furthermore, rare G418-selected Huh-7
clones "cured" of Con1 replicons that had not acquired adaptive mutations during
the selection process were more permissive for HCV replication than "cured" cells
originally supporting adapted Con1 subgenomic RNA replication (Blight et al.,
2002b). These results demonstrate that cellular environments more conducive to
HCV replication are ultimately selected and, in Huh-7 cells that harbor conditions
most supportive for viral replication, transfected Con1 replicon RNA does not need
to adapt to efficiently replicate.

The most permissive "cured" subline identified so far (Huh-7.5; Blight et al., 2002b)
has the capacity to support high levels of subgenomic HCV replication in >75%
of transfected cells. Furthermore, Huh-7.5 cells more readily support RNAs with
lower replicative abilities, such as full-length Con1 replicons (Blight et al., 2002b)
and H77-derived RNAs (Blight et al., 2003). Increased permissiveness in Huh-7.5
cells is due to mutational inactivation of the retinoic acid inducible gene-I (RIG-I), a
cytoplasmic protein that recognizes structured RNA to induce type I IFN production
via activation of transcription factors interferon regulatory factor (IRF)-3 and NF-
κΒ. Complementation with functional RIG-I restores IRF-3 signaling in Huh-7.5
cells and converts this hyper-permissive cell line to a relatively non-permissive
phenotype (Sumpter Jr. et al., 2005). Thus, RIG-I-mediated activation of IRF-3 is
a critical determinant of cellular permissiveness for HCV replication.

ADDITIONAL CELL LINES

Attempts by many investigators to propagate HCV RNAs in other cell lines were
unsuccessful until 2003, when Zhu and coworkers described replication of Con1
subgenomic RNAs in HeLa cells and in the murine hepatoma cell line Hepa1-
6, albeit with low efficiency (Zhu et al., 2003). Due to the high error rate of the
NS5B RdRp, replicon RNAs prepared from Huh-7 cell lines in which persistent
replication had been established were expected to have greater genetic variance
than HCV RNAs generated by in vitro transcription from cloned cDNA templates.
Indeed, transfection of HeLa cells with total RNA from Con1 replicon-harboring
Huh-7 cells gave rise to a low number of G418-resistant cell colonies. Replicons
in G418-resistant HeLa cells maintained the Huh-7 cell adaptive mutations, but

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acquired several additional mutations in the non-structural proteins. Interestingly,

the colony-forming efficiency of replicon RNAs isolated from HeLa cells was
significantly higher in naïve HeLa cells than Huh-7 cells. In addition, HeLa cell-
derived replicon RNAs were more efficient at establishing G418-resistance in
HeLa cells than replicons obtained from Huh-7 cells. These results indicate that
replicon variants have been selected that can replicate more efficiently in HeLa
cells. However, in vitro transcribed subgenomic RNAs carrying both the Huh-7 and
HeLa cell-specific mutations only conferred G418 resistance in a few HeLa cells,
and thus the relative contribution of these mutations to productive replication in
HeLa cells has not been determined. Replicon RNAs derived from HeLa cells, but
not from Huh-7 cells, were also able to replicate and confer resistance to G418 in
a few Hepa1-6 cells. Sequence analysis revealed that replicons from these mouse
cells had preserved the majority of the mutations found in HeLa cells, and only a
few additional mutations were identified. Moreover, total RNA isolated from one
of the Hepa1-6 replicon-containing cell lines did not increase the colony-forming
efficiency compared to replicon RNA from HeLa cells, suggesting that HeLa-
derived subgenomes were already adapted for replication in the mouse cells (Zhu
et al., 2003).

Con1 replicon replication has also been established in human embryonic kidney 293
cells. By co-culturing replicon-containing Huh-7 cells with 293 cells, rare hybrids
closely resembling parental 293 cells were selected that supported subgenomic RNA
replication (Ali et al., 2004). Nucleotide sequence analysis of replicating HCV RNA
in hybrid 293 cells identified a large number of mutations that appear to facilitate
replication in 293 cells; transfection of total cellular RNA isolated from one of these
replicon-expressing hybrid clones was able to establish replication in naïve 293 cells.
As observed by Zhu and coworkers (Zhu et al., 2003), in vitro transcribed replicon
RNA, containing all the mutations identified in this hybrid 293 clone, failed to confer
resistance to G418 in naïve 293 cells (Ali et al., 2004). The differences in colony-
forming capacity between in vitro transcribed Con1 subgenomic RNA and replicon
RNA isolated from stable cell clones remains a mystery, although it is possible
that RNA molecules co-purifying with the replicating replicon RNA facilitates the
establishment of Con1 replication in these less permissive cell lines.

Although in vitro transcripts derived from genotype 1b replicons have not

successfully established replication in non-Huh-7 cells (Zhu et al., 2003; Ali et al.,
2004), G418 resistance in human hepatocyte- (HepG2 and IMY-N9; Date et al.,
2004) and non-hepatocyte- (HeLa and 293; Kato et al., 2005) derived cell lines
has been achieved by the genotype 2a replicon, JFH-1. The efficiency of colony
formation was 10- to 1000-fold lower compared to Huh-7 cells (Date et al., 2004;
Kato et al., 2005), and the colony-forming efficiency and colony size in HepG2 and
IMY-N9 cells was increased following transfection of JFH-1 replicons harboring

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the Huh-7 specific adaptive mutation in NS5B (H2496L; Date et al., 2004). Many
G418-selected cell clones did not contain mutations in the HCV coding region
and, when coding changes were found, they were not shared between independent
clones and had not been previously identified in JFH-1 replicons replicating in
Huh-7 cells (Date et al., 2004; Kato et al., 2005). Interestingly, in eight of the nine
293-derived cell clones analyzed, amino acid substitutions were not identified (Kato
et al., 2005). These findings suggest that adaptive mutations are not essential for
JFH-1 replication in either hepatocyte- or non-hepatocyte-derived cell lines.

Collectively, the cell tropism for HCV genotypes 1b and 2a has not only been
expanded to include additional human hepatoma cell lines, but also non-liver derived
human cells and murine hepatocytes, disproving the previous hypothesis that HCV
replication is governed by hepatocyte- and primate-specific factors. Moreover, the
ability of HCV replicons to replicate in a murine hepatoma cell line offers some
hope that a mouse model for HCV infection may be developed in the future.

APPLICATIONS OF THE HCV REPLICON SYSTEM

Since the introduction of HCV replicons in 1999 and the subsequent identification
of adaptive mutations a year later, numerous researchers have utilized the replicon
system to probe the mechanisms of HCV replication, define the roles of individual
proteins, identify the viral and cellular determinants of HCV replication, and
examine the interplay between HCV and the Huh-7 cell. This section provides
specific examples to highlight the utility of this cell culture model to address these
fundamental questions about HCV biology.

The availability of stable cell lines that harbor autonomously replicating subgenomic
RNAs has facilitated the study of viral protein expression, subcellular localization
of HCV replication, and the structure, function, and biochemical properties of the
replication complex. Additionally, the mechanisms by which HCV counteracts the
host antiviral response, the effects of HCV replication on host cell function and
the importance of host factors for efficient replication have begun to be unraveled.
The HCV polyprotein is proteolytically processed in a preferential order with rapid
cleavages at the NS3/4A and NS5A/5B sites, while the NS4A-4B-5A precursor is
processed at a slower rate (Pietschmann et al., 2001), confirming previous studies
using heterologous expression systems (Lin et al., 1994; Bartenschlager et al.,
1994; Tanji et al., 1994). The mature HCV proteins have half-lives ranging from
10 to 16 hours, except the hyperphosphorylated form of NS5A, which appears
less stable (Pietschmann et al., 2001). Similar to all positive-sense RNA viruses
investigated so far (reviewed in Ahlquist et al., 2003; Salonen et al., 2005), HCV
reorganizes intracellular membranes to form a membranous web. This altered
membrane represents a site of HCV replication in Huh-7 cells (Gosert et al., 2003),
and by utilizing the replicon encoding the NS5A-GFP fusion (Fig. 3B), active HCV

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Blight and Norgard

replication complexes have been directly visualized in living cells by fluorescence

microscopy (Moradpour et al., 2004b). Interestingly, fluorescence is strongest
in subconfluent cells, consistent with earlier studies showing that subgenomic
RNA replication is strongly influenced by the proliferation status of the cells with
replication stimulated in the S phase of the cell cycle, but rapidly declining in
confluent or serum-starved cells (Pietschmann et al., 2001; Scholle et al., 2004).
The ability to track functional replication complexes should aid in defining the
steps involved in membranous web formation and in the assembly and turnover of
replication complexes. Although the origin of the membranous web has not been
defined, its close proximity to the ER and the ER localization of the non-structural
proteins in replicon-containing cells (Pietschmann et al., 2001; Mottola et al., 2002;
El-Hage and Luo, 2003; Miyanari et al., 2003) suggest the web is derived from
membranes of the ER. In contrast, the association of HCV RNA and non-structural
proteins with NP40-insoluble membranes in replicon-containing cells and their
cofractionation with caveolin-2 suggest that active replication complexes reside
on lipid rafts recruited from intracellular sites (Shi et al., 2003; Gao et al., 2004;
Aizaki et al., 2004). Clearly more experimentation is required to define the origin
of the membranes on which the HCV replication complex assembles.

HCV subgenomic replicon-containing Huh-7 cells also provide a source of

membrane fractions containing crude replication complexes for biochemical studies
(Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). These complexes retain
enzymatic activity as evidenced by the synthesis of replicon-length RNA from the
endogenous (co-fractionating) template RNA. Furthermore, partially single-stranded
and double-stranded replicative forms are synthesized, transcription of single- and
double-stranded RNAs are differentially effected by Mg2+ and Mn2+, RNA synthesis
is actinomycin D-resistant, and de novo initiation of RNA transcription occurs in
these isolated membrane fractions. Thus, the use of enzymatically active HCV
replication complexes rather than employing NS5B alone offers an in vitro system to
probe the structure and function of the replicase and to evaluate potential inhibitors
targeting RNA replication. Interestingly, these crude replication complexes have
failed to utilize exogenously added template RNA (Ali et al., 2002; Hardy et
al., 2003; Lai et al., 2003) and most of the viral RNA in these membrane-bound
complexes is nuclease resistant (El-Hage and Luo, 2003; Miyanari et al., 2003;
Yang et al., 2004), consistent with the notion that HCV replication complexes
are relatively closed structures. Furthermore, treatment of these complexes with
proteinase K degrades more than 90% of the viral proteins with no effect on RNA
transcription (Miyanari et al., 2003), thus only a minor fraction of HCV proteins
are engaged in RNA synthesis and this fraction is protected within the replication
complex.

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 HCV Replicons

Like many viral infections, HCV triggers the host cell antiviral response in part
through the accumulation of replication intermediates or the presence of double-
stranded RNA structures within the HCV genome (Pflugheber et al., 2002; Wang
et al., 2003; Sumpter Jr. et al., 2005). Persistent HCV infections frequently develop
(Alter and Seeff, 2000), suggesting that HCV has evolved efficient mechanisms to
counteract the intracellular antiviral response. These mechanisms are beginning to be
elucidated using stable cell lines supporting persistent subgenomic HCV replication.
The protease action of the HCV NS3/4A complex has been shown to disrupt two
independent signaling pathways, toll-like receptor 3 (TLR3) and RIG-I, that both
induce type I IFN production (Li et al., 2005; Sumpter Jr. et al., 2005; Breiman et
al., 2005). While the protease target in the RIG-I signaling pathway has yet to be
identified (Sumpter Jr. et al., 2005; Breiman et al., 2005), the adaptor protein (toll-
IL-1 receptor domain-containing adaptor inducing IFN-β; TRIF) linking TLR3 to the
kinases responsible for activating the latent transcription factors, IRF-3 and NF-κB,
is proteolytically cleaved (Li et al., 2005). Additionally, NS5A has been implicated
in the ability of HCV to block the host response to double-stranded RNA. Direct
binding of NS5A with protein kinase R (PKR) disrupts signaling events that activate
IRF-1 (Pflugheber et al., 2002) or limit RNA translation (Wang et al., 2003).

Inhibition of host factors either through RNA interference or expression of

dominant-negative mutants of the protein has facilitated the identification of host cell
factors important for productive HCV replication. For example, by these strategies
polypyrimidine tract binding protein (PTB; Zhang et al., 2004; Domitrovich et al.,
2005), La autoantigen (Zhang et al., 2004; Domitrovich et al., 2005), and human
vesicle-associated membrane transport protein A (hVAP-A; Gao et al., 2004; Zhang
et al., 2004) have been found to be important for HCV replication. Furthermore,
hVAP-A interacts with NS5B (Gao et al., 2004) and NS5A (Evans et al., 2004b; Gao
et al., 2004) and recently it has been suggested that NS5A hyperphosphorylation
disrupts interactions between hVAP-A and NS5A to negatively regulate HCV
replication (Evans et al., 2004b).

Reverse genetics in the replicon system has become an important tool to define HCV
RNA sequences and protein determinants critical for productive RNA replication
in Huh-7 cells. For instance, the first 125 nucleotides of the 5' NTR are sufficient
for RNA replication, demonstrating that the regions required for translation and
replication overlap (Friebe et al., 2001; Kim et al., 2002; Reusken et al., 2003; Luo
et al., 2003). Mapping studies have been conducted on the 3' NTR and confirm the
observations made in chimpanzees experimentally inoculated with RNA transcripts
carrying similar mutations in the 3' NTR (Kolykhalov et al., 2000; Yanagi et al.,
1999). The terminal 98 nucleotides and poly(U/UC) tract are indispensable for
replication, although the poly(U/UC) region can be shortened without affecting HCV
replication (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003). In contrast,
the upstream variable region can be deleted, resulting in a 100-fold reduction in

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Blight and Norgard

G418-resistant colonies (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003).

More recently, critical cis-acting replication sequences in addition to the 5' and 3'
NTRs have been identified. Mutational disruption of a computer-predicted highly
conserved stem-loop structure located within the 3' terminal coding region of NS5B,
designated 5BSL3.2, blocks subgenomic replication (Lee et al., 2004a; You et al.,
2004; Friebe et al., 2005) and can be restored when an intact copy of this RNA
element is inserted into the 3' NTR (Friebe et al., 2005). Furthermore, 5BSL3.2
and the terminal 98 nucleotides form a pseudoknot structure that is indispensable
for HCV replication (Friebe et al., 2005).

As mentioned above, HCV replication occurs in conjunction with rearranged

membranes, and thus it is not surprising that most of the HCV non-structural
proteins contain membrane-anchoring segments (NS4A, 4B, 5A, and 5B; reviewed
in Dubuisson et al., 2002; Moradpour et al., 2003). Membrane association of
NS5A and NS5B, mediated by an N-terminal amphipathic helix in NS5A (Elazar
et al., 2003) and the C-terminal 21 amino acid residues of NS5B (Moradpour et
al., 2004a; Lee et al., 2004b), is essential for productive HCV replication in Huh-7
cells. Although the determinants for membrane association have been defined and
their importance in HCV replication is beginning to be recognized, the composition
and interactions required to assemble functional replication complexes are poorly
understood. Nonetheless, sequences that may mediate interactions essential for
replicase assembly are being identified. For instance, solvent-exposed residues
in the N-terminal helix of NS5A (Penin et al., 2004) and sequences within the
transmembrane region of NS5B (Ivashkina et al., 2002; Moradpour et al., 2004a;
Lee et al., 2004b) appear to play critical, but as yet undefined, roles in subgenomic
replication beyond those of membrane insertion. Two regions of NS5A previously
reported to mediate interactions with NS5B in vitro (Shirota et al., 2002) have been
shown to be important for productive HCV replication (Shimakami et al., 2004).

Finally, by applying reverse genetics in the replicon system, a GTP-binding motif

(Einav et al., 2004) and an amphipathic helix (Elazar et al., 2004) in NS4B as well
as a conserved zinc-binding motif in the N-terminal domain of NS5A (Tellinghuisen
et al., 2004) have been found to be important for efficient HCV replication, although
at this stage the exact role of these motifs in replication has not been determined.
As discussed above, hyperphosphorylated NS5A is detrimental to HCV replication,
while basal phosphorylation of NS5A has recently been shown to be nonessential
for productive HCV replication in Huh-7 cells (Appel et al., 2005b). Thus, further
experimentation is warranted to determine whether phosphorylation of NS5A is
required at all for RNA replication or whether it serves a regulatory purpose, such
as signaling the switch between RNA replication and virus particle assembly. As
illustrated above, subgenomic replicons provide an excellent model system for
examining HCV-host interactions and for molecular studies of HCV replication.

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 HCV Replicons

INHIBITION OF REPLICON REPLICATION

With the development of HCV replicons it became possible to analyze the role of
cytokines in the cellular defense against HCV. Genotype 1 replication in cell culture
is sensitive to the antiviral programs induced by IFN-α (Blight et al., 2000; Frese
et al., 2001; Guo et al., 2001; Gu et al., 2003; Kato et al., 2003a; Lanford et al.,
2003; Tanabe et al., 2004; Kanda et al., 2004; Ikeda et al., 2005), IFN-β (Kato et al.,
2003a; Larkin et al., 2003), IFN-γ (Frese et al., 2002; Kato et al., 2003a; Lanford et
al., 2003; Larkin et al., 2003), IFN-λ (Robek et al., 2005), and interleukin-1 (IL-1;
Zhu and Liu, 2003), but not tumor necrosis factor-α (Lanford et al., 2003). In all
cases examined so far, the 50% inhibitory concentrations (IC50) for IFN-α in Huh-7
cells are very low (0.5-3 IU/ml; Gu et al., 2003; Tanabe et al., 2004; Ikeda et al.,
2005) and are independent of the ISDR (Blight et al., 2000). Type I IFNs induce the
transcription of a large number of genes that encode effector proteins with antiviral
activities, including PKR, 2'-5' oligoadenylate synthase (OAS), IFN-stimulated
gene 56 (ISG56), and MxA guanosine triphosphatase. Although MxA inhibits the
replication of a broad variety of RNA viruses (Haller and Kochs, 2002), it appears
that IFN activity in Huh-7 cells proceeds via a MxA-independent pathway since
expression of MxA does not inhibit HCV replication and expression of a dominant-
negative MxA does not interfere with the antiviral effects of IFN (Frese et al.,
2001). Other studies have suggested that IFN-α blocks HCV replication through
translational control involving PKR (Wang et al., 2003) and ISG56 (Sumpter Jr. et
al., 2004). Thus, the underlying mechanisms of antiviral activity of type I IFNs as
well as IFN-γ, IFN-λ, and IL-1 in the replicon system remain unresolved.

Ribavirin significantly improves the rate of sustained viral clearance compared to

monotherapy with IFN-α (McHutchison et al., 1998; Di Bisceglie and Hoofnagle,
2002). Similarly, ribavirin and IFN-α in combination elicit strong synergistic
inhibitory effects on subgenomic HCV replication (Tanabe et al., 2004; Kanda
et al., 2004). The underlying therapeutic mechanism of ribavirin is unknown,
but may involve induction of lethal mutagenesis, inhibition of RdRp activity,
depletion of intracellular nucleotide pools via inhibition of the host enzyme inosine
monophosphate dehydrogenase (IMPDH), or stimulation of the cellular immune
response. In Huh-7 cells, ribavirin exhibits antiviral activity by acting as an RNA
mutagen inducing error-prone HCV replication (Lanford et al., 2003; Zhou et
al., 2003; Tanabe et al., 2004; Kanda et al., 2004). On the other hand, exogenous
guanosine can suppress the mutagenic effect of ribavirin and potent IMPDH
inhibitors enhance the antiviral capacity of ribavirin, suggesting that ribavirin can
inhibit HCV replication by depleting GTP pools (Zhou et al., 2003). The HCV
replicon system is being utilized to generate ribavirin-resistant variants. Interestingly,
two conserved mutations in the C-terminal region of NS5A independently confer
a low level of ribavirin resistance, while ribavirin-resistant HCV replication
could also be attributed to defects in ribavirin import rather than mutations in the

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replicon RNA (Pfeiffer and Kirkegaard, 2005). Thus, HCV replicons will continue
to provide a valuable model system for elucidating the mechanisms of ribavirin
action, synergism with IFN, and ribavirin resistance in cultured cells.

Subgenomic replicons encode all the known cis-acting RNA sequences and viral
enzymes (Table 1) required for replication that are now prime targets for antiviral
drug design. Thus, the replicon system provides an excellent screening platform
to identify compounds that effectively block enzymatic activities of HCV-encoded
proteins and to evaluate the inhibitory effect of nucleic-acid based approaches
including antisense oligonucleotides, ribozymes, and small interfering RNAs
(siRNAs). Small-molecule inhibitors of the HCV NS3/4A serine protease that are
effective in the nanomolar range have been identified (Lamarre et al., 2003; Pause et
al., 2003) and nucleoside analogues and non-nucleoside small molecules have been
explored as RdRp inhibitors (Carroll et al., 2003; Tomei et al., 2004; Beaulieu et al.,
2004; Ludmerer et al., 2005; Tomassini et al., 2005). A peptidomimetic inhibitor
of the protease, BILN 2061, provided the first proof-of-principle for preclinical
evaluation of new antiviral drugs using the replicon system. BILN 2061 was very
active at inhibiting genotype 1 subgenomic replication (IC50 3-4 nmol/L; Lamarre
et al., 2003) and in a phase 1 clinical trial, BILN 2061 administered orally to HCV
genotype 1-infected patients led to a 100-1000-fold drop in circulating virus within
two days of treatment (Lamarre et al., 2003; Hinrichsen et al., 2004). In contrast,
only half of the patients persistently infected with HCV genotype 2 or 3 who
received BILN 2061 for 48 hours responded with a reduction in viral RNA greater
than 1 log10 (Reiser et al., 2005), underscoring the need to develop replicon-based
screening assays for the remaining HCV genotypes (genotypes 3, 4, 5 and 6).
Another major factor limiting the efficacy of therapies to combat HCV infection
will be the ability of HCV to develop resistance to specific antiviral drugs, but
the HCV replicon system will allow potential drug-resistant variants to be rapidly
identified and characterized. For example, drug-resistant substitutions in the NS3
protease domain emerge when replicon-containing cells are cultured in the presence
of active inhibitors of the HCV protease (Trozzi et al., 2003; Lin et al., 2004; Lu et
al., 2004) and a single mutation within the NS5B polymerase conferred resistance
to a nucleoside analog, although replicons carrying this mutation were attenuated
(Migliaccio et al., 2003). Replicon-containing cells are also being used to test the
efficacy of RNA interference against HCV RNAs. Synthetic or stably expressed
siRNAs and small hairpin RNAs targeting various regions of the HCV non-structural
coding sequence (NS3, NS4B, and NS5B) as well as the 5' NTR efficiently suppress
HCV replication, albeit with different efficiencies (Randall et al., 2003; Yokota et
al., 2003; Seo et al., 2003; Wilson et al., 2003; Kapadia et al., 2004; Kronke et al.,
2004; Takigawa et al., 2004). Interestingly, siRNAs are more effective at reducing
HCV RNA levels than high doses (100 IU/ml) of IFN-α (Kapadia et al., 2004).
Furthermore, replicating RNA can be cleared from >98% of siRNA-transfected

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cells (Randall et al., 2003), and siRNAs, introduced into Huh-7 cells prior to HCV
replicons, effectively prevent the establishment of HCV replication (Wilson et al.,
2003). Thus, these studies support the principle of siRNA-based HCV antiviral
therapy; however, the challenges that have hindered nucleic acid therapies in
the past still need to be resolved. As we have already begun to witness, the HCV
replicon system will be fundamental for determining the antiviral potency of HCV
inhibitors, optimizing drug regimens, monitoring for drug-resistance, and assessing
the efficacy of nucleic-acid based antiviral strategies.

CONCLUDING REMARKS
The development of subgenomic replicons capable of autonomous replication in
the human hepatoma cell line Huh-7 marked an important turning point for HCV
research. The identification of cell culture-adaptive mutations and highly permissive
Huh-7 sublines has enabled the development of transient replication assays as
well as replication-competent monocistronic subgenomes, replicons that encode
reporter genes, and full-length HCV genomes. Replicons derived from isolates
belonging to genotype 1a, 1b, and 2a are now available and the cell tropism for
HCV genotypes 1b and 2a has been expanded to other hepatoma cell lines, non-
liver-derived cells, and murine hepatocytes. For the first time, HCV replication can
be studied at the molecular level in cell culture. Many investigators have capitalized
on this system to investigate important questions related to HCV biology that have
plagued the field since the molecular cloning of the HCV genome 16 years ago.
While significant advances have been made, there are many questions that remain
which undoubtedly will be answered by future research. For instance, what are the
mechanisms underlying cell culture adaptation of genotype 1 isolates? Why does
the JFH-1 genotype 2a sequence not require adaptive mutations to replicate in
cell culture? What are the factors that facilitate more efficient HCV replication in
Huh-7 cells than in the other cell lines tested? Why do adaptive mutations prevent
virus particle assembly? The recent discovery that full-length RNAs encoding the
unmodified non-structural proteins from genotype 2a JFH-1 produce infectious virus
particles overcomes one of the remaining barriers and now allows the complete
viral life cycle to be studied in cell culture. Finally, subgenomic RNAs are already
proving valuable for the development and evaluation of antiviral drugs as well
as screening for the emergence of drug resistance. The replicon system should
accelerate the development of effective drugs to cure individuals chronically
infected with HCV.

ACKNOWLEDGMENTS
We are grateful to Dan Ader, John Majors, Sondra Schlesinger, and Milton
Schlesinger for critical reading of the manuscript. Supported in part by the Ellison
Medical Foundation New Scholars in Global Infectious Disease Research Program to
K.J.B. (ID-NS-0119-03). E.A.N. is supported by the Monticello College Foundation

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Olin Fellowship for Women. We apologize to colleagues for the omission of other
key literature citations due to space limitations.

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Chapter 12

Animal Models for HCV Study

Linda B. Couto and Alexander A. Kolykhalov

ABSTRACT
The study of HCV biology is complicated by the paucity of relevant animal models.
The ideal model for studying HCV would be one that adequately represents most
aspects of human HCV infection and disease, is affordable, easily available, and
reproducible. Currently, the only widely recognized animal model of HCV infection
is the chimpanzee, which does not meet all of these desirable attributes. Recently,
other models have been used to dissect various aspects of HCV biology and to
evaluate novel therapeutics. Each has a unique set of advantages and limitations.
Transgenic mouse models have elucidated the pathophysiology of specific viral
proteins, but they are limited by their inability to support HCV replication. Xenograft
models provide an environment for human hepatocyte engraftment in mice and
subsequent infection with HCV. These models are technically challenging, but
once optimized they promise to be extremely useful both for the study of HCV
biology and for drug development. Alternatively, the GBV-B virus, which efficiently
replicates in tamarins and marmosets, represents a surrogate model for the study
of HCV. Chimeras between GBV-B and HCV have been created and will be useful
in the development of HCV-targeting drugs.

CHIMPANZEE MODEL
Use of the chimpanzee model to address questions regarding HCV biology is well
justified by both the importance of HCV as a major healthcare problem and by the
fact that the questions cannot be addressed otherwise. The chimpanzee is the closest
genetic relative to human, which explains why many features of hepatitis C disease
are so common between humans and chimps. Both humans and chimpanzees have
detectable HCV RNA within a few days of infection. Maximum viral titers usually
reach 105-107 RNA genome copies per mL of blood. The rise in viremia is usually
followed by an increase in serum liver enzymes, which peak between 2 and 12
weeks. The majority of infected chimpanzees have necroinflammatory changes
in liver biopsies; typically the disease is somewhat milder than that observed in
humans. Antibodies to HCV antigens usually appear around week 8 or after. This
acute phase of infection is followed by transition toward chronic viral persistence. It
was initially reported that chimpanzees have lower rates of chronicity compared to
humans, ~40% and ~70-85%, respectively. Bassett et al. (1998) reported that a cross-

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sectional study in the chimp colony at the Southwest Foundation for Biomedical
Research, San Antonio, TX, revealed that out of 46 animals infected with different
strains of HCV, only 18 (39%) were viremic based on reverse transcription-PCR
analysis. More recent data on infection of naïve animals with HCV suggest that
about 60% of all chimps became persistently infected; this rate of persistence is
similar for HCV of different genotypes (Bigger et al., 2004; Nam et al., 2004).
Chimpanzees with acute resolving infection usually clear virus in plasma during
weeks 12 to 24.

As the only animal model for the study of HCV, the chimpanzee was used to
provide early characteristics of HCV. Even before HCV had been identified, the
chimpanzee was involved in the study of Non-A, Non-B hepatitis (NANBH) virus
transmission, in establishment and duration of disease, and in the chronic nature of
NANBH infection (Alter et al., 1978; Tabor et al., 1978). The first characteristics
of the infectious agent, such as its size (Watanabe et al., 1987), and its inactivation
with lipid solvents (i.e. the enveloped nature of the agent) (Bradley et al., 1983;
Feinstone et al., 1983) were determined using the chimpanzees. Finally, the HCV
genome was isolated and cloned for the first time from chimpanzee plasma with
a high infectivity titer of Non-A, Non-B agent (Bradley et al., 1991; Choo et al.,
1989).

The major advantages of the chimpanzee model stem from the ability to monitor
and analyze the development of the disease from its initiation. Most clinical data
on HCV infection in humans is derived from patients who have been infected for
a period of time, often decades. Due to the asymptomatic nature of hepatitis C
disease, the acute phase of the infection is often not noticeable, and thus, very little
data exist regarding the events immediately following infection. In human studies,
usually only samples of easily accessible tissues, such as blood, are available. Only
a few liver biopsies per year can be performed in infected patients, which preclude
efficient analysis of events in the primary tissue of HCV replication. On the contrary,
liver biopsy samples from the chimpanzees can be obtained before the exposure
and at planned intervals post-inoculation. These well controlled samples allowed
the analysis of events starting immediately after HCV infection, such as changes
in gene regulation and cellular immune responses to viral antigens. Furthermore,
the possibility to rechallenge animals that cleared a previous infection, allowed
memory immune responses to be analyzed (Bassett et al., 2001; Bigger et al., 2004;
Farci et al., 1992; Ilan et al., 2002b; Prince et al., 1992; Weiner et al., 2001), as
well as an analysis of HCV vaccine development (Forns et al., 2000; Houghton,
2000). Finally, the chimpanzee model was instrumental in the establishment of
HCV infectious molecular clones (Kolykhalov et al., 1997; Yanagi et al., 1997). The
virus recovered from an in vitro synthesized RNA resulted in the development in
chimpanzees of classical signs of hepatitis disease, such as viremia, elevated serum

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levels of hepatic enzymes, histologic changes in the liver, and the development of
HCV specific antibodies, thus formally proving that HCV is the causative agent
of the disease.

REVERSE GENETICS/FUNCTIONAL ANALYSIS OF HCV

The ability to test the infectivity of molecular clones in chimpanzees allowed for
the first time a reverse genetics analysis. This established the critical importance of
all genome coded enzymatic activities, as well as some cis-acting elements in the
HCV genome, for virus replication (Kolykhalov et al., 2000; Bukh et al., 1999).
Though the HCV replicon tissue culture model is extremely useful for genetic
analysis, it is restricted by the fact that replication of HCV RNA is not dependent
on structural proteins (see Chapter 11). Thus, the chimpanzee model was required
to demonstrate that the hypervariable region 1 (HVR1) of the envelope protein E2
is not critical for virus replication in vivo, and can be removed altogether (Forns et
al., 2000). This result was somewhat unexpected since the HVR1 was considered
among the primary regions of HCV to interact with the host immune system (Farci
et al., 1996; Kato et al., 1993). In another experiment, it was demonstrated that p7
is absolutely essential for infectivity of HCV, and that the amino- and/or carboxyl-
terminal intraluminal tails of p7 contain sequences with genotype-specific function
(Sakai et al., 2003).

MONOCLONAL INFECTIONS
Many experiments using patient-derived virus were complicated by the fact that
HCV exists as a set of quasispecies. Replication of the viral genome depends on
the genome-encoded RNA dependent RNA polymerase, which lacks proofreading
activity (see Chapter 10). As a consequence, viral replication results in the
accumulation of numerous genetic variants, called quasispecies. The quasispecies
nature of an inocula was thought to explain the initial evolution of the virus in
vivo, as well as the escape of the virus from the immune response (Hijikata et al.,
1995; Kojima et al., 1994; Okamoto et al., 1992). Recovery of virus from in vitro
synthesized infectious RNA allowed the creation of "monoclonal" virus pools,
derived from a single cDNA molecule. Chimpanzee serum, collected during the
first weeks following intrahepatic inoculation of infectious RNA, exhibited no
genetic variability (Major et al., 1999), therefore, providing a unique starting
material for the study of viral evolution and of virus-host interactions. Infection of
chimpanzees with such virus simplifies studies of HCV, since the interpretation of
results is not complicated by the quasispecies nature of the inocula. Thus, Major
at al. (2004) published detailed results of the analysis of ten chimps all inoculated
with the same monoclonal virus, representing the dominant variant in the most
studied patient isolate H77. Six out of ten infected animals became chronically
infected, which implied that the presence of quasispecies in the inocula is not a
requirement for establishing chronicity. The acute phase of infection was similar in
all animals, whether they resolved the infection or became chronically infected. The

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maximal viral titers were 0.5-1 log higher in animals with chronic infection, usually
between 106 and 107 RNA copies/ml. The viral load increased quickly during the
first 1-2 weeks after infection (mean doubling time = 0.5 days) until reaching 103
to 105 copies/ml. This was followed by a significant delay in virus accumulation
(mean doubling time = 7.5 days) over the next several weeks, during which titers
increased by only 2 to 3 log10. Viral titers began to decrease in all animals as alanine
amino transferase (ALT) responses increased, with peak RNA titers preceding ALT
peaks by 2 to 3 weeks. ALT elevations in the serum are believed to be markers of
hepatocyte death and could be due to killing of infected or bystander liver cells
by the host immune response. Both the height and the time of the ALT peaks were
similar between the groups. Following the ALT peak the viral titers decreased, and
all infections resulted in 1 of 2 outcomes: persistence, with virus titers reaching a
steady state at approximately 104 to 105 RNA copies/mL; or clearance, with titers
continuing to decrease below levels of quantitation (<200 copies/mL in the study).
After the decrease in viral titers, ALT levels returned to baseline in all animals
despite significant levels of virus in those animals with persistent infection. The
animals that became persistently infected were followed for 82-216 weeks after
infection. Very low levels of the virus were observed in some animals after the
clearance from time to time up to 1 year. This was attributed to the use of a highly
sensitive RT-PCR method for virus detection (40 RNA copies/mL) and to very long
follow-up of the cleared animals.

An HCV-specific antibody response in the Major at al. (2004) study was mounted
during weeks 7-14 (usually on weeks 9-10) in the chronic group and during weeks
6-9 (majority on weeks 8-9) in the animals that resolved the infection. On the
contrary, antibodies to the HVR1 were detectable only in the chronic group (in 5
out of 6 animals), but not in the resolved group. In general, anti-HVR1 antibodies
correlate with anti-E1/E2 antibodies (Bartosch et al., 2003; Major et al., 1999). In
an overlapping study by Logvinov et al. (2004) no neutralizing antibody (nAb)
responses were detected in three animals that cleared the virus, whereas strain-
specific nAbs were detected in six of the seven chronically infected animals after
approximately 50 weeks of infection. These data suggest that nAbs do not play
a role in the control of virus infection. This data correlates between human and
chimpanzee (Prince et al., 1999).

CELLULAR IMMUNE RESPONSE

The role of the cellular immune response was also addressed in the chimpanzee
model. The early events in a human infection are difficult to analyze, since liver
tissue samples are not available during the acute phase of infection, nor are control
liver samples taken from before the infection. Analysis of T-cell responses showed
that animals who terminated the infection mounted a strong cytotoxic T lymphocyte
(CTL) response (Cooper et al., 1999; Nascimbeni et al., 2003), and that CD8+ CTLs

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are better correlated with protection against HCV infection than antibodies. The
appearance of these cells in the liver several weeks after infection was temporary
associated with increases in liver enzymes in the plasma and with a temporary
decrease in viral load in plasma . Thimme at al. (2002) showed that initial HCV
spread outpaces the T cell response by demonstrating that HCV rapidly induces, but
is not controlled by IFN-alpha and IFN-beta. Viral clearance follows the appearance
and accumulation of HCV-specific IFN-γ-producing T cells in the liver. The
importance of memory CD8+ T cells in the control of HCV infection was confirmed
by antibody-mediated depletion of this lymphocyte subset in a chimpanzee (who
had recovered from two previous infections) just before a third infection with the
same dose and strain of HCV. Virus replication was significantly prolonged despite
the presence of memory CD4+ T helper cells primed by the two prior infections,
and it was not terminated until HCV-specific CD8+ T cells recovered in the liver
(Shoukry et al., 2003). This was in sharp contrast to the second infection, when the
effector function was not delayed, and the viremia was terminated within 14 days,
i.e. 28 days earlier than during the third infection. Control of this second infection
was kinetically linked to the rapid acquisition of virus-specific cytolytic activity by
liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in
the blood. Though memory CD4+ T cells were intact in the CD8+ T cell-depleted
animals, they did not facilitate rapid clearance of the virus. It does not, of course,
rule out a critical supporting role for the helper T cells in protective immunity.
This was addressed in other two chimpanzees that had cleared the infection. These
animals were treated with an anti-CD4 monoclonal antibody before reinfection
with HCV (Grakoui et al., 2003). The treatment resulted in significant reduction
of circulating CD4+ T cells for over one year, that, in turn, resulted in persistent,
low-level viremia despite functional intra-hepatic memory CD8+ T cell responses.
Incomplete control of HCV replication by memory CD8+ T cells in the absence of
adequate CD4+ T cell help was associated with emergence of viral escape mutations
in class I major histocompatibility complex (MHC)-restricted epitopes and in the
failure to resolve HCV infection.

Most studies of T-cell mediated immunity to HCV suggest that this response is
essential for resolution of infection. However some animals with high numbers
of intrahepatic CD4+ and CD8+ T cells did not clear the infection completely. In
these animals, a decrease of viral titers during the acute phase was followed by viral
persistence. One explanation of this fact was provided by Erickson et al.(Erickson
et al., 2001), who showed that HCV in three persistently infected chimpanzees
acquired mutations in multiple epitopes that impaired class I MHC binding and/or
CTL recognition. Most escape mutations appeared during acute infection and
remained fixed in the viral population for years without further diversification. A
statistically significant increase in the amino acid replacement rate was observed
in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes

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were intact in animals that resolved hepatitis C spontaneously. Other explanations

for the inefficiency of the HCV-specific CD8+ T cells include the possibility that at
least a portion of the cells is anergic or arrested at an early stage of differentiation
(Gruener et al., 2001; Ulsenheimer et al., 2003; Wedemeyer et al., 2002), that
infected hepatocytes are resistant to immune recognition, and/or that HCV-specific
CD8+ T repressor cells are present that produce anti-inflammatory cytokines, such
as IL-10 (Accapezzato et al., 2004).

Prediction of the outcome of an HCV infection is complicated by the fact that

despite the well documented correlation between the spontaneous resolution of
infection and the presence of a vigorous cellular response, this correlation is not
absolute. In the study by Thompson et al. (2003), it was shown that neither the
chimpanzees that remained chronically infected, nor the animals that resolved the
infection mounted a significant cellular response. Only weak and transient T helper
responses were detected during the acute phase in all animals. This study suggests
that chimpanzees may recover from HCV infection by mechanisms other than the
induction of readily detectable HCV-specific T-cell responses.

LIVER GENE EXPRESSION

Changes in liver gene expression in response to HCV infection were measured
in acute phase samples of chimpanzees who either became chronically infected,
temporarily controlled the infection, or cleared the infection (Bigger et al., 2001; Ilan
et al., 2002b). In another study, chronic phase samples were compared for changes
in gene expression in the livers of 10 chimpanzees (Bigger et al., 2004). Hundreds of
genes were shown to be up or down regulated in response to HCV infection. Some
changes in the expression profile are expected, such as changes due to the response
to viral double-stranded (ds) RNA, which includes type 1 interferons (IFNs) and
the IFN response genes. Changes due to the innate and adaptive immune response
to the infection are also predicted, including activation and infiltration of NK cells,
macrophages, and lymphocytes. In addition, changes due to the hepatocyte response
to the cytokines expressed by the immune cells are expected. To determine the set
of genes whose expression most likely reflects the initial host response to HCV in
the liver, Su et al. (2002) attempted to identify genes with expression patterns that
strongly correlated with the amount of HCV RNA in the serum of all of the chimps
over the entire time course profiled, and found 27 such transcripts. Many of these
genes were known to be stimulated by IFN-α, including STAT1, 2'-5' oligoadenylate
synthase (OAS), and Mx1, which are well known to exert antiviral activity through
inhibition of translation, activation and repression of transcriptional activity, and
mRNA degradation (de Veer et al., 2001). Similarly, Bigger et al. (2001) found
that during the first phase of infection the most notable changes in gene expression
occurred in numerous IFN response genes that increased dramatically, some as
early as day 2 post-infection. Moreover, Su et al. (2002) showed that although

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HCV-infected cells successfully induce the transcription of many antiviral IFN-

α-stimulated genes, this response has little or no effect on viral titer or outcome.
Analysis of gene expression in 10 chronically infected chimpanzees confirmed that
many IFN-stimulated genes were transcriptionally elevated, suggesting an ongoing
response to IFN and/or dsRNA (Bigger et al., 2004). On the contrary, transient and
sustained viral clearance was uniquely associated with the induction of IFN-γ-
induced genes and other genes involved in antigen processing and presentation and
the adaptive immune response (Su et al., 2002). IFN-γ-induced genes are known to
be expressed as a result of the homing and activation of immune cells to the liver
(Boehm et al., 1997). Surprisingly, induction of IFN-γ itself was not detected in
Su et al. (2002) microarray analysis, probably due to its low expression levels. The
increases in IFN-γ mRNA levels were detected by RT-PCR in all tested animals in
the Major at al. (2004) study, and these increases coincided with ALT elevations
and decreases in viral titers in the plasma. In the cohort of animals infected with the
same monoclonal virus, the induction of IFN-γ was observed both in those animals
with self-limiting infections and in chronically infected animals. Additionally, the
level of induction did not correlate with spontaneous clearance of the virus; IFN- γ
induction was at least as high in the group of animals that progressed to chronic
infections as for those that eventually cleared HCV. Also, the levels of IFN- γ mRNA
remained elevated into the post-acute phase to a similar degree in all animals. Out
of four genes reported in this study for which data were collected, two genes had
profiles of the expression that correlated with the outcome; induction of the CD3e
and MIP-1a were observed only in animals that cleared the virus. The initial peaks
of CD3 also coincided with the control of virus replication.

A single genotype 3-infected animal was available for analysis in study performed
by Bigger et al. (2004), and this animal exhibited increased expression of a number
of genes potentially involved in steatosis compared to the levels of expression in
animals with genotype 1 infections.

Unlike human patients, for whom the timing of infection, route of infection, the
source and nature of the virus are often not known, the chimpanzee provides a
well controlled clinically relevant model for the study of HCV. However, it is
extremely limited in its availability, as well as by its expense. As a consequence,
many results have been generated in experiments with low numbers of animals. In
order to generate more statistically credible data, alternative animal models that
are readily available and less expensive are needed.

TRANSGENIC MOUSE MODELS

A number of transgenic mouse models have been developed to examine the potential
pathogenic effects of the HCV core protein and/or the envelope glycoproteins
on hepatocytes. Conflicting results have been reported, and therefore it has been

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hard to make any conclusive statements regarding the pathogenicity of the HCV
structural proteins. In one study, the core protein of HCV (genotype 1a) was shown
to be produced in mouse liver at levels similar to that seen in chronically infected
HCV individuals, and pathogenesis was not observed over the course of 18 months
(Pasquinelli et al., 1997). However, in another similar transgenic line expressing the
core protein from HCV (genotype 1b), vacuolations in the liver were observed which
led to steatosis in animals at 3-12 months of age (Moriya et al., 1997). Steatosis is
the abnormal accumulation of fat within hepatocytes, and it appears to be a factor
affecting chronic hepatitis C progression in humans (Rubbia-Brandt et al., 2004).
In follow-up studies, Moriya et al. (1998) reported the formation of gross hepatic
nodules in animals at 16 months of age, and the development of hepatocellular
carcinoma (HCC) in some animals, suggesting that the core protein plays an
important role in HCC. In addition, an age dependent increase in oxidative stress
within the liver, as measured by an increase in lipid peroxidation and a decrease
in glutathione levels, was observed in the transgenic mice that developed HCC
(Moriya et al., 2001). As endogenous oxidants are an important class of naturally
occurring carcinogens that act by producing genetic alterations, this may contribute
to the etiology of HCC. In a transgenic line that was created to express not only
core, but also E1 and E2 of HCV (genotype 1b), hepatitis was observed at 10-15
months, but no neoplastic nodules or carcinomas were observed during 4 years of
cumulative observation in animals that ranged in age from 4-20 months.(Honda et
al., 1999). Another transgenic line expressing these same three transgenes showed
no evidence of liver pathology during the six months these animals were evaluated
(Kawamura et al., 1997). In addition, no histological abnormalities associated with
the expression of the envelope proteins alone were observed in transgenic mice up
to 18 months of age (Koike et al., 1995; Pasquinelli et al., 1997). The conflicting
results regarding the pathology associated with the HCV structural proteins could
be due to differences in the mouse strains used to generate the transgenic animals.
Alternatively, the discrepancies could be attributed to the different promoters that
were used to express the transgenes, which resulted in different levels of protein.

One of the drawbacks of using transgenic models to study the potential pathogenesis
of HCV proteins is the fact that the animals are tolerant to the transgenic protein,
and thus, the role of the immune response to HCV proteins cannot be evaluated.
To overcome this limitation, Wakita et al. (1998) constructed a transgenic model
that allows conditional expression of the core, E1,E2, and NS2 proteins of HCV
(genotype 1b) using the Cre/loxP recombination system. An adenoviral vector
expressing Cre DNA recombinase was used to induce the expression of the HCV
proteins. Upon infection of these mice with the adenoviral vector, acute hepatitis was
observed and a humoral response to core protein was detected, indicating that the
transgenic animals were immunocompetent for HCV proteins. To determine the role
of the cellular immune response in the development of hepatitis, CD4+ and CD8+

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T cells were depleted by administration of anti-CD4 and anti-CD8 monoclonal

antibodies. In the absence of T cells, no histopathological differences were observed
between Ad-infected and uninfected transgenic mice, suggesting that HCV structural
proteins are not directly cytopathic to hepatocytes, but rather a cellular immune
response to these proteins is responsible for the hepatitis observed. However, a
caveat to this interpretation is that adenoviral vectors alone cause hepatitis in a T
cell-dependent manner, so it is not clear if the hepatitis observed was caused by
the HCV structural proteins or the adenovirus (Yang et al., 1996). In a follow-up
study (Wakita et al., 2000), these authors reported that injection of the Ad-Cre
vector increased the CD8+ lymphocyte infiltration in the livers of transgenic mice
more than that in non-transgenic mice, and ALT levels were higher in the former.
In addition, CTLs isolated from the livers of transgenic mice were HCV specific,
suggesting that HCV structural proteins are indirectly responsible for liver injury,
and that the host immune response plays a role in the pathogenesis of HCV. The
Cre/loxP system is a useful model to evaluate host/viral protein interactions, but
it could be improved by expressing the Cre protein from a non-inflammatory viral
vector, such as an adeno-associated viral vector.

Although still controversial, HCV infection has been reported to be associated

with several extrahepatic manifestations, including hypertrophic and dilated
cardiomyopathy. Using transgenic mice expressing the HCV core protein, Omura et
al. (2005) recently demonstrated the development of histological changes consistent
with cardiomyopathy after 12 months of age. However, the pathogenicity of these
cardiac complications is not well understood.

XENOGRAFT MODELS
Two xenograft models for studying HCV have been developed and are now being
used to evaluate HCV biology and anti-HCV therapies. Both models rely on
transplantation of human hepatocytes into mice and subsequent repopulation of the
mouse liver. One model utilizes Alb-uPA transgenic mice, which carry a tandem
array of four murine urokinase-type plasminogen activator genes under the control
of a liver-specific albumin promoter (Heckel et al., 1990). Over-expression of the
transgene is cytotoxic to hepatocytes and results in a hypofibrinogenemic state
and fatal neonatal bleeding. Spontaneous inactivation of the transgene occurs in a
portion of the cells, giving them a growth advantage over transgene-containing cells,
and the uPA-negative cells eventually repopulate up to 90% the liver (Sandgren et
al., 1991). Mercer et al. (2001) combined the properties of these mice with those
of immunodeficient SCID mice to develop a model system that allows human
hepatocyte engraftment in mouse liver, and used this as a small animal model to
study HCV infection. In this model transgenic uPA mice are transplanted with a
million human hepatocytes, and engraftment occurs over the course of 4-6 weeks.
Following engraftment the animals are inoculated with HCV-infected serum from

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human donors. Evidence for replication of HCV in these animals was confirmed
by demonstrating viral titers of 1x104-1x106 copies/ml for periods up to 35 weeks.
In addition, the authors claimed to detect negative-strand RNA, supporting the
contention that bona fide HCV viral replication occurs in the animals. Viral
replication was further validated by serially passaging the virus through three
generations of mice, during which viral RNA levels increased 37,500 times.
Considering dilution of the virus during passaging, this could not be attributed to
the original human inoculum. These results clearly demonstrate that both replication
of the HCV genome and production of fully infectious viral particles is possible
in this animal model.

Recently, this chimeric mouse/human model was used to evaluate novel anti-HCV
therapies. In one report (Hsu et al., 2003), the mice were used in a gene therapy
approach to treat HCV by delivering a modified form of the BH3-interacting death
agonist (BID). BID is a member of the Bcl-2 family of pro-apoptotic proteins and
is crucial for death receptor-mediated apoptosis (Esposti, 2002). It is activated
upon cleavage by caspase 8, and induces an increase in the permeability of the
outer mitochondrial membrane, leading to release of apoptogenic proteins, such as
cytochrome c. In this study, the endogenous cleavage site of BID was engineered to
contain a specific cleavage site recognized by the NS3/NS4A protease of HCV. An
adenoviral vector encoding the modified BID was delivered to the livers of SCID/
Alb-uPA mice that had been previously infected with HCV. Animals were evaluated
for serum HCV RNA titers, as well as, clinical and liver pathology. HCV-infected
animals had initial HCV titers that ranged from 1x104-5x107 genome equivalents/ml.
The animals with lower titers were able to completely clear the infection following
Ad-BID vector administration, while a 2-3 log decrease in HCV viral titers was
observed in animals with higher initial titers. Histological examination of the livers
of animals inoculated with both HCV and Ad-BID showed extensive cell death,
and a TUNEL assay confirmed apoptosis in their livers.

A company called KMT Hepatech (Edmonton, Alberta) has now been founded on
the basis of this chimeric human/mouse technology, that provides "KMT mice" to
collaborators interested in evaluating potential HCV therapies. Although not yet
published in a peer-reviewed journal, a scientific poster on the company's website
details the use of this model to evaluate two anti-HCV therapeutics that have been
used successfully to treat HCV in humans (P-187 10th HCV Meeting Dec2-6,2003
Kyoto, Japan; KMT Hepatech website). The two drugs tested were IFN-α-2B and
the novel protease inhibitor, BILN2061, which has been shown in human clinical
trials to reduce viral RNA levels by 2-3 logs (Lamarre et al., 2003). Homozygous
SCID/Alb-uPA mice transplanted with human hepatocytes were treated with either
IFN-α-2B or BILN2061, and statistically significant reductions in HCV viral
loads were observed with both agents. Thus, two therapies that have been shown

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to be effective against HCV in humans, and one novel gene therapy, are able to
reduce viral titers in HCV-infected "KMT mice", thereby validating the model for
evaluation of novel anti-HCV therapies.

Another mouse model that has been developed to study HCV replication is a
modification of the "Trimera mouse". Trimera mice are created by total body
irradiation of normal mice, followed by reconstitution with bone marrow from
SCID mice. These animals are subsequently engrafted with human hematopoietic
cells or solid tissues, such as liver. Since the resulting animal is comprised of three
genetically distinct sources of tissue, the name Trimera mouse was coined. These
mice were originally created to study the development of human B and T cells, but
they are now also used to study HCV. When human liver fragments are transplanted,
engraftment rates of up to 85% have been reported one month post-implantation
(Ilan et al., 2002a). When healthy human liver tissue fragments were infected ex vivo
with HCV positive serum, viremia was detected in 50% of transplanted animals.
Mean viral loads of up to 1x105 copies /ml peaked on day 18, and subsequently
declined by day 25. Direct transplantation of infected human livers also resulted in
viremia in mice for approximately one month. The decline in viremia is the result
of fibrosis and necrosis of the human liver tissue, which limits the evaluation of
potential anti-HCV therapeutics. In addition to the presence of viral RNA in the
serum of mice, HCV RNA was also detected in the implanted human liver tissue.
Whereas positive-strand RNA was observed on day 0, negative-strand RNA was not
observed until day 9, suggesting that HCV replication occurs. The trimeric model
has been shown to support replication of HCV 1a, 1b, 2a, and 3a.

Having established that bona fide HCV replication occurs in the Trimera mouse
model, it was used to evaluate the efficacy of two novel anti-HCV agents. A small
molecule HCV IRES inhibitor and an anti-HCV monoclonal antibody showed
modest dose-dependent reductions in the viral load of HCV during the treatment
period, which returned to pretreatment levels following cessation of drug treatment
(Ilan et al., 2002a). Viability of the hepatocytes was assessed by measuring the
levels of human serum albumin (HSA) mRNA in grafts from control and treatment
groups. Similar levels of HSA mRNA were observed, demonstrating that the
reduced viral load was not due to a hepatotoxic effect of the drug. In these studies
the maximal initial viral load was approximately 7x104 copies/ml. This level of
viremia will need to improve before potent anti-viral agents, that have the ability
to knock down HCV titers by several logs can be tested. Another issue with the
model is that viremia persists for only one month, limiting the timeframe available
to test the efficacy of drugs. At present, protocols to increase this window using
antifibrotic agents are being attempted.

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Maeda et al. (2004) recently proposed an alternative to the Trimera model,

which involves engrafting human liver tissue into non-obese diabetic/severe
combined immunodeficiency (NOD/SCID) mice. Although very good engraftment
(approximately 90%) was observed in these mice, inoculation of these animals with
either HCV-infected human serum or culture media containing an infectious HCV
molecular clone resulted in HCV viral titers that were near the limit of detection of
the PCR-based assay. HCV sequences were detected in the engrafted liver tissue
by in situ PCR, but at present this does not represent a robust animal model for
studying HCV replication or for evaluating anti-HCV therapies.

All xenograft models that have been described rely on RT-PCR for quantification
of HCV RNA. To reliably detect changes in titer, the viral load should be at least
10,000 copies/ml, in order to see efficacy in the range of a one log decrease in
titer. The main advantage of these models is that they represent a potentially less
expensive in vivo model for studying HCV relative to the chimpanzee. Another
advantage of the xenograft models is that HCV infection and replication occurs
in human hepatocytes as opposed to chimpanzee or other non-human primate
hepatocytes. Compared to the tissue culture replicon systems, adaptive mutations
in the HCV genome do not seem to be required for replication in the xenograft
models. However, none of these models has yet to achieve widespread utility due to
the technical difficulty in breeding and creating the mice, the limited availability of
human hepatocytes, variable human cell engraftment and inconsistent viral titers. If
these difficulties can be overcome, these models will greatly aid the study of HCV
biology and pathogenesis, as well as facilitate the development of new therapies
for HCV. In order to expand the application of these mouse models for studying
the role of the immune system in the pathogenesis of HCV, it will be necessary to
reconstitute the mice with components of the human immune system.

SMALL NON-HUMAN PRIMATE MODELS

The chimpamzee is the only truly validated animal model for studying HCV, but
because they are an endangered species, expensive to work with, and the subject
of ethical debates, other non-human primates were evaluated for infection by HCV.
Early studies concluded that NANBH virus could infect marmosets (Feinstone et
al., 1981) and tamarins (Karayiannis et al., 1983), both of which are New World
monkeys. However, these studies were undertaken before the NANBH virus group
had been subdivided into identifiable agents and before specific diagnostic tests for
HCV were available. Once these were obtained, it was concluded that chimpanzees,
but no other non-human primates, were susceptible to HCV infection (Garson et
al., 1997).

Despite this, the pursuit of a small-animal model to study HCV has not abated,
and the GBV-B virus, which efficiently replicates in tamarins and marmosets, is an

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example of a surrogate model of HCV that has been gaining credibility. Originally
identified as a "GB hepatitis agent", it was transmitted to tamarins from the blood
sample of a surgeon (whose initials are GB) that was suffering from acute hepatitis
(Deinhardt et al., 1967). All infected tamarins developed an acute hepatitis, and
subsequently, the GB hepatitis agent was identified as containing two distinct RNA
viruses: GBV-A and GBV-B (Simons et al., 1995). It was later shown that GBV-A
does not replicate in the tamarin liver, whereas GBV-B causes hepatitis. Sequence
analysis of the GBV-A and GBV-B genomes suggested that they belong to the
Flaviviridae family. The GBV-B virus contains a positive-sense, single-stranded
RNA genome of 9399 nucleotides, and it was shown to be most closely related
to HCV. Additional experimental infection of tamarins using either the original
serum sample, or serially passaged infected tamarin serum, confirmed that GBV-B
causes acute hepatitis (Beames et al., 2000; Bright et al., 2004; Bukh et al., 1999).
In three different species of tamarins, viremia with peak viral titers in excess of
1x108 genomic equivalents/ml was observed between 2 and 14 weeks post-injection,
which subsequently cleared by 16 weeks post-injection. Viremia was accompanied
by an increase in liver enzymes and inflammation in the liver. Because tamarins,
like chimpanzees, are in limited supply, another New World monkey, the common
marmoset, has been assessed for susceptibility to GBV-B infection. Marmosets
are easier to manage as breeding colonies and are currently bred for biomedical
research in a number of facilities. Several reports have confirmed the susceptibility
of marmosets to GBV-B infection (Bright et al., 2004; Jacob et al., 2004; Lanford et
al., 2003). Viral titers of over 1x108 genome equivalents/ml peaked around 6 week
post-injection and viral clearance was complete by week 16. Increases in some
liver enzymes were observed, which correlated with inflammation in the liver, as
the result of infiltration of CD8+ lymphocytes. This GBV-B marmoset model was
recently tested as a small-animal model for HCV by evaluating several anti-HCV
therapeutics. A small-molecule inhibitor of the HCV NS3 protease was also shown
to inhibit GBV-B replication in vivo (Bright et al., 2004). This inhibitor reduced
GBV-B viral replication by more than three logs. This was the first time an anti-
HCV therapeutic was demonstrated to be effective in an animal model other than the
chimpanzee, and provides validation of the GBV-B/marmoset model as a surrogate
for studying HCV. Not only is this model valuable for evaluating therapies directed
against the NS3 protease, it may soon be shown to be useful for testing therapies
targeting other regions of the HCV genome.

One of the major differences between the course of infection of GBV-B in New
World monkeys to that of HCV in humans is the tendency for the former to result
in an acute infection and the propensity of the latter to lead to chronic hepatitis.
However, in one recent study viremia was observed for >2 yrs following infection
of one tamarin with GBV-B (Martin et al., 2003). In this case, the animal was
infected by direct intrahepatic inoculation of synthetic RNA. It is not clear if this

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was related to the outcome, but in any case, it enhances the value of the GBV-B
tamarin or marmoset model.

The cloning and sequencing of GBV-B revealed some differences and similarities
between GBV-B and HCV (Muerhoff et al., 1995) and allowed the construction
of chimeric viruses to be made between the two. The genomic organization and
structure of GBV-B and HCV are similar; each containing a single long ORF
flanked by 5' and 3' nontranslated regions (NTR). The 5' portion of the ORF was
predicted to encode structural proteins, while the 3' portion of the ORF encodes
nonstructural proteins (Muerhoff et al., 1995). Even though the homology of the
predicted polyproteins between GBV-B and HCV is low (25-30%), the hydropathy
plots of the polyproteins are very similar. The early work that led to the realization
of chimeric viruses was the demonstration that the GBV-B and HCV NS3 protease
share substrate specificity (Scarselli et al., 1997). This guided the construction of
a functional chimeric GBV-B/HCV protease that consisted of an N-terminal HCV
protease domain and a C terminal GBV-B RNA helicase domain. The chimeric NS3
retained protease activity capable of processing both GBV-B and HCV substrates,
and retained helicase activity similar to that of the native GBV-B NS3 (Butkiewicz
et al., 2000). The ability to construct a chimeric NS3 polypeptide with HCV
protease and GBV-B helicase activities suggested that it may be possible to create
viable chimeric GBV-B/HCV viruses that could be used to test protease inhibitors
in the tamarin and/or marmoset model. For this to be realized, the development
of an infectious clone of GBV-B was required. Although the GBV-B genome was
initially cloned and sequenced in 1995, RNA transcribed from this clone was not
infectious. It was not until 1999, when it was shown that the 3'NTR extends an
additional 259 nucleotides, that an infectious clone was generated (Bukh et al.,
1999). This set the stage for the construction and evaluation of chimeric viruses.
This area is currently in its infancy and chimeric viruses may eventually serve as
models for testing HCV protease, helicase, or polymerase inhibitors, as well as,
therapeutic agents that target the viral RNA.

A recent publication (Rijnbrand et al., 2005) describes a functional chimeric virus

derived from GBV-B, in which a functionally important HCV regulatory sequence
was substituted for the analogous sequence in the 5' NTR of GBV-B genome.
Domain III of the GBV-B NTR, which binds directly to the 40S ribosome subunit,
was replaced with the corresponding domain from HCV. Inoculation of tamarins
with RNA transcripts derived from this chimeric clone led to recovery of viable
virus, which resulted in acute hepatitis. This result demonstrates that domain III
of HCV can substitute for the similar domain in GBV-B and can support both
translation and viral replication in vivo. However, the kinetics of viremia were
noticeably different, and the unusual infection profile was shown to be due to
the accumulation of compensatory mutations that arose to support efficient viral

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replication. This is an exciting new model for the evaluation of HCV replication
and for use in drug screening, as this chimeric virus will allow the investigation of
potential anti-HCV therapies targeted to domain III of the HCV IRES.

Similar chimeras may soon be available in the 3'NTR region of GBV-B, following
the functional analysis of mutant 3'NTR sequences. The GBV-B 3'NTR consists of a
short sequence of 27 nucleotides, followed by a poly(U) tract of 23 nucleotides, and
a 3' terminal sequence that consists of 309 nucleotides. By deleting specific regions
of the 3'NTR and testing the mutants by intrahepatic transfection of tamarins with
the transcribed RNAs, functionally important areas of the 3'NTR were identified.
Deletion of both the poly (U) tract and the short proximal sequence killed the virus;
however, deletion of just one of these elements resulted in viable viruses (Nam et
al., 2004). The authors also showed that insertion of a long heterologous sequence
in the proximal sequence resulted in the recovery of a virus that had deleted the
majority of the insert, but retained a short fragment of the heterologous sequence.
This suggests that it should be possible to insert short DNA sequences into the
GBV-B 3'NTR.

Studies using chimeric replicating viruses in the marmoset model, while providing
a new modality for testing anti-HCV therapies, will certainly have limitations.
These viruses may not completely mimic the HCV viral life cycle and the insertion
of HCV elements into the GBV-B sequence may not accurately reflect the natural
accessibility of these elements in the HCV genome. However, it represents a new
small-animal model alternative to the chimpanzee and will be useful in evaluating
at least some potential drug candidates.

SUMMARY AND CONCLUSIONS

The chimpanzee remains the best animal model for studying the biology of HCV, as
it is the only animal that is susceptible to HCV infection and replication, and because
the liver disease observed in chimpanzees mimics the pathology seen in humans.
Nevertheless, this model is difficult to use due to its expense, inaccessibility, and
ethical considerations, and thus, efforts to develop other smaller animal models
have continued. A major advantage of a small-animal model is that large numbers of
animals can be employed, thus, providing statistically significant data. A few mouse
models have been in the development now for about one decade. Transgenic mice
expressing HCV structural proteins, for example, can be used to detect potential
pathophysiological features of specific viral proteins. However, these animals cannot
be used to probe questions about the viral life cycle. In addition, in all but one
transgenic model, the mice are tolerant to the HCV proteins preventing the immune
response against these proteins from being evaluated. In one model, conditional
expression of HCV proteins is possible. Since the host immune response to HCV
is believed to play an important role in disease, this latter model may be very

367
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useful. Several mouse/human xenograft models are also being used to study HCV
biology and to evaluate potential anti-HCV therapeutics. These models, although
very promising, still suffer from a lack of reproducibility and require skilled and
experienced individuals to create them. Another drawback, is that the animals are
immunocompromised, and thus virus/host interactions cannot be assessed fully.
Finally, the use of GBV-B/HCV chimeric viruses that infect and replicate in New
World monkeys offer the advantages of direct virus infection of a small animal,
without the complication and irreproducibility of hepatocyte engraftment. Although
these viruses may not perfectly recapitulate HCV biology, drugs that target specific
regions of these chimeric viruses can be evaluated using this model. All of the
animal models for studying HCV have their limitations, but careful selection of a
model will allow investigators to ask specific questions regarding HCV infection,
replication, pathogenesis, and/or drug sensitivity, and important information can
be gleaned.

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a single full-length cDNA clone of hepatitis C virus are infectious when directly
transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 8738-
8743.

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Chapter 13

HCV Regulation of Host Defense

D. Spencer Carney and Michael Gale Jr.

ABSTRACT
Mammalian cells respond to virus challenge by initiating a "host response"
characterized by interferon α/β (IFN) production and a cellular antiviral state. The
host response is our first line of immune defense against viral pathogens and it
imposes several barriers that hepatitis C virus (HCV) must overcome to replicate and
persist. HCV evades the host response through a complex combination of virus-host
interactions that disrupt intracellular signaling pathways and attenuate the antiviral
actions of IFN. Regulation of the host response breaks a link between innate and
adaptive immunity and provides a foundation for HCV replication and spread.

INTRODUCTION
Exposure to HCV typically leads to persistent infection associated with a chronic
disease course. The ability of HCV to mediate persistent, life-long infection in its
human host is linked to the evasive nature of the virus to thwart the host immune
system and to resist the antiviral actions of IFN-based therapy. Molecular studies
of HCV-host interactions have revealed several levels of immune regulation and
evasion directed by HCV protein products. This chapter provides an overview of
the virus-host interface of these regulatory processes and their impact on HCV
replication and persistence.

INNATE INTRACELLULAR IMMUNE DEFENSES

In response to virus infection, signaling pathways within mammalian cells direct
a variety of intracellular events that generate an antiviral state directly within the
infected cell. This antiviral response, termed the 'host response" to virus infection,
represents our first line of immune defense against virus infection. If this response
is successful, exposure to the virus will render a self-limiting, abortive infection.
It is the hepatic host response that imposes initial immune defenses against HCV
infection (Gale, Jr., 2003). The host response is triggered when the infected
cell recognizes a molecular signature within the invading virus. This signature,
known as a pathogen-associated molecular pattern (PAMP), is typically a physical
characteristic of the virus that is recognized and engaged by specific PAMP receptor
proteins within the host cell (O'Neill, 2004). The PAMP/PAMP receptor interaction
initiates signaling cascades that induce the expression of antiviral effector genes

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Fig. 1. Triggering the host response to HCV infection. HCV triggers the host response through
the process pathogen-associated molecular pattern (PAMP)/PAMP receptor engagement. Certain
RNA motifs within the HCV genome have PAMP attributes that activate the host response. RIG-I
(Sumpter et al., 2005) and possibly TLR3 confer HCV PAMP recognition. Signaling through these
and possibly other pathways leads to the activation of IRF-3, IFN production and ISG expression.
Autocrine and paracrine signaling by IFN can potentiate viral PAMP signaling and serves to amplify
the host response through a feedback loop involving IRF-7 and PAMP signaling molecules. IFNs
also enhance immune cell maturation and effector function, resulting in a host response aimed at
controlling virus infection.

(Sen, 2001). For RNA viruses, protein and nucleic acid products of infection
comprise an array of PAMP signatures that can engage specific PAMP receptors,
including Toll-like receptors (TLRs) and nucleic acid binding proteins (Fig. 1;
Iwasaki and Medzhitov, 2004; Cook et al., 2004). The HCV RNA contains specific
PAMP signatures, including poly-uridine motifs and stem-loop double-stranded
RNA (dsRNA) structures within its single-stranded RNA genome (Tuplin et al.,
2002; Simmonds et al., 2004). HCV RNA is sufficient to induce the host response
in cultured hepatocyte-derived cell lines (McCormick et al., 2004; Sumpter et
al., 2005). The product of retinoic acid inducible gene I (RIG-I), which has been
defined as a dsRNA PAMP receptor, is critical for host response signaling induced
by HCV RNA (Yoneyama et al., 2004; Sumpter et al., 2005). In hepatocytes, the
independent signaling pathways of RIG-I and Toll-like receptor 3 (TLR3) direct
the host response to virus infection (Li et al., 2005a).

PAMP/PAMP receptor engagement signals the activation of a variety of transcription

factors in the infected hepatocyte. PAMP-driven transcription factor activation
results in the immediate expression of host response genes (Fig. 2; Malmgaard,
2004). Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB)
are triggered in response to virus infection, and each are activated upon cellular
recognition of the HCV PAMP (Au et al., 1995; Lin et al., 1999; Fredericksen
et al., 2001; Richmond, 2002; Prabhu et al., 2004). Their activation proceeds
through PAMP-responsive signaling pathways of the cell that promote their nuclear
translocation and transactivation functions. Other IRF family members, including
IRF-5 and IRF-7, are essential components of the host response to virus infection
(Barnes et al., 2001; Kawai et al., 2004), though the specific role of each in HCV
infection has not been characterized. Virus-induced signaling events that activate
ATF-2 and further direct chromatin remodeling contribute to the building of a virus-
triggered enhancesome on the IFN-β promoter. The IFN-β enhanceosome includes

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Fig. 2. Signaling the host response to HCV infection. 1. Viral PAMP (HCV RNA) binding to RIG-I ,
TLR3 or other PAMP receptors results in the phosphorylation and activation of IRF-3 by the TBK1
or IKKε protein kinases. The dimer of phospho-IRF-3 translocates to the cell nucleus, interacts with
its transcription partners (Yoneyama et al., 1998), and binds to the cognate DNA positive regulatory
domain (PRD) in the promoter region of IRF-3 target genes, including IFN-β. 2. IRF-3 activation
drives IFN-β production. 3. IFN-β binding to the IFN α/β receptor signals the activation of the
associated Tyk2 and Jak1 protein kinases and the phosphorylation/ assembly of a STAT1/STAT2
heterodimer and trimeric ISGF3 (Sen, 2001). 4. ISGs are induced by ISGF3. ISGs are the genetic
effectors of the host response, and IRF-7 is itself and ISG and a critical transcription factor whose
actions are turned on through viral PAMP signaling pathways that overlap with the pathways of
IRF-3 activation. IRF-7 phosphorylation, dimerization and heterodimerization with IRF-3 directs its
binding to the virus-responsive element (VRE) in the promoter region of IFN-α genes, resulting in
IFN-α subtypes expression (Au et al., 2001). This increases the abundance of RIG-I and viral PAMP
signaling components to amplify host response. The therapeutic administration of IFN-α provides
antiviral action against HCV by signaling ISG expression through the IFN α/β receptor and the Jak-
STAT pathway. The NS3/4A protease prevents IFN-β production by disrupting RIG-I signaling and
cleaving TRIF to ablate TLR3 signaling (Foy et al., 2005; Li et al., 2005b).

IRF-3 and NF-κB, which produce a transcriptional response resulting in IFN-β

expression and secretion from the infected cell (Sen, 2001). NF-κB activation and
function is central to the chemokine and proinflammatory cytokine response to virus
infection, which functions side by side with IFN to modulate the ensuing adaptive
immune response (Tai et al., 2000). Secreted levels of IFN-β drive autocrine and
paracrine signaling processes by binding the IFN-α/β receptors of the infected cell

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and local surrounding tissue, respectively. This results in activation of the Jak-STAT
pathway. Here, receptor-associated Jak and Tyk1 protein kinases phosphorylate
signal transducer and activator of transcription (STAT) proteins on critical serine
and tyrosine residues to confer STAT activation, STAT association with IRF-9, and
nuclear localization of the resulting ISGF3 transcription factor complex. ISGF3 is
the central transcription factor that promotes high level expression of the interferon
stimulated genes (ISGs) by binding to the IFN-stimulated response element (ISRE)
within the ISG promoter/enhancer region. IFN binding to the IFN α/β receptor and
signaling of the Jak-STAT pathway drives a second wave of transcriptional activity
initiated by virus infection and denoted by the expression of ISGs.

ISG PRODUCTS HAVE ANTIVIRAL FUNCTIONS

The human genome encodes hundreds of ISGs (Der et al., 1998). The ISG products
direct regulatory functions that control virus infection. ISGs have been shown
to interrupt HCV replication through processes that include suppression of viral
protein synthesis and synthesis inhibition of the viral negative strand replicative
intermediate (Guo et al., 2001; Shimazaki et al., 2002; Wang et al., 2002; Prabhu
et al., 2004). The main feature resulting from the secretion of IFN from infected
cells into the local tissue is the paracrine induction of a tissue-wide host response
that blocks cell to cell spread of the virus. Many components of the host response
pathways are themselves ISGs, and though expressed basally at a low level that
facilitates surveillance and response to virus infection, their abundance will increase
in response to IFN signaling. In the liver, paracrine signaling of IFN serves to
enhance overall responsiveness of cellular signaling pathways to potentiate the
host response to infection in a tissue-wide manner. IFN signaling thus provides an
amplification loop to further promote the host response against HCV (Foy et al.,
2005). Recent studies have shown that the amplification loop is dependent upon
the transcriptional activity of IRF-7 (Honda et al., 2005). IRF-7 is an ISG and its
expression in the liver is IFN-dependent (Smith et al., 2003; Honda et al., 2005). IRF-
7 promotes the expression of the various IFN-α subtypes, wherein IFN-α production
and secretion mediate further amplification of the host response and prolonged IFN
production (Honda et al., 2005). IFN-based therapy for HCV infection exploits
these actions of IFN-α to limit HCV replication and spread (Fig. 2) (McHutchison
and Patel, 2002). IFN-α also signals the maturation of immune effector cells,
antigen presenting cells and dendritic cells, and it potentiates the production of
other proinflammatory cytokines by resident hepatic cells to indirectly modulate
cell-mediated defenses and adaptive immunity (Biron, 1999). Viral triggering and
control of the host response may therefore define cellular permissiveness for HCV
RNA replication and influence the outcome of infection.

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CROSS-TALK BETWEEN INNATE AND ADAPTIVE IMMUNE

DEFENSES.
IFN-α is a potent immunomodulator that influences the onset of the cellular immune
response and adaptive immunity to HCV infection. IFN-α modulates natural
killer (NK) cell activity toward lysis of infected target cells by promoting NK cell
activation and proliferation, and supporting their survival through the induction
of IL-15 production (Biron, 1999; Loza and Perussia, 2004). Activated NK cells
produce IFN-γ (Shoukry et al., 2004). In the HCV-infected liver, NK cell homing and
local secretion of IFN-γ may serve to limit HCV replication directly. Indeed, IFN-γ
has been shown to mediate direct antiviral effects against HCV RNA replication in
vitro (Frese et al., 2002), and its production by immune effector cells in the liver
associates with viral clearance in the chimpanzee model of HCV infection (Thimme
et al., 2001; Su et al., 2002). IFN-α also influences the maturation of dendritic cells
and modulates their presentation of viral antigens (Colonna et al., 2004; Barth et
al., 2005). Antigen presentation by dendritic cells under the influence of IFN-α
contributes to the differentiation of CD4 T cells toward the Th1 phenotype and
importantly, a Th1-predominate response is associated with clearance of HCV
infection (Shoukry et al., 2004). IFN-α production during HCV infection may
therefore indirectly contribute toward directing the maturation of CD4 T cells to
the Th1 phenotype. Dendritic cells are also involved in the cross priming and IFN-γ
production of CD8 T cells. In this context the co-stimulatory signals presented at
the time of antigen cross presentation to CD8 T cells can determine whether the
cells are cross primed for a cytotoxic response or cross-tolerized for anergy (Cooper
et al., 1999; Shoukry et al., 2004). TLR3 plays an essential role in cross priming
by dendritic cells, in which it signals the production of IFN-α triggered by viral
PAMPs within products of phagocytosis (Schulz et al., 2005). IFN-α produced by
the dendritic cell in this manner induces the expression of co-stimulatory molecules
and cytokines to promote the cross priming and activation of CD8 cells, thus linking
host response signaling processes to induction of the adaptive immune response.
The link between innate, IFN-induced antiviral pathways and the adaptive immune
response is not entirely understood and further studies are required to define the
linkage of these processes with the outcome of HCV infection.

IFN-γ ACTIVATION OF THE ANTIVIRAL RESPONSE

IFN-γ may serve to complete the signaling loop between infected hepatocytes
and immune effector cells. The IFN-γ receptor is expressed by hepatocytes and
in most other tissues. In the liver, IFN-γ produced by infiltrating NK cells and
activated T cells, where it can bind its receptor in paracrine fashion to induce a
hepatic IFN-γ response. IFN-γ receptor binding leads to phosphorylation of STAT1
through the receptor-bound Jak1 and Jak2 protein kinases (Stark et al., 1998). The
phosphorylated STAT then forms a homodimer that translocates to the cell nucleus
and acts on the gamma-activated sequence elements of target genes to promote their

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transcription (Der et al., 1998). In hepatocytes the genes under the control of the
GAS element have significant overlap with those expressed in response to IFN α/β
and under control of the ISRE (Cheney et al., 2002). GAS elements are also found
within genes whose products are involved in antigen processing and presentation,
immune effector action and apoptosis, thus deriving a variety of antiviral actions.
A role for GAS element genes in host defense against HCV is supported by studies
of the chimpanzee model for HCV infection, where the high expression of IFN-γ
responsive genes in the liver has associated with the resolution of acute infection
in the chimpanzee model (Su et al., 2002). Together with IFN-α, the actions of
IFN-γ provide addition levels of host defense cross-talk that serves to limit HCV
infection.

HEPATIC DEFENSES ARE TRIGGERED BY HCV

Functional genomic analyses from cohorts of patients infected with HCV have
shown that infection associates with a hepatic gene expression profile marked by
ISGs whose expression levels vary widely among patients (Smith et al., 2003). These
observations indicate that HCV can induce and regulate the hepatic host response to
infection. Gene profiling studies of HCV-infected chimpanzees have demonstrated
that acute resolving HCV infection is associated with a host response characterized
by high level hepatic ISG expression (Bigger et al., 2001). In similar studies,
"outcome predictor" gene sets were identified by the overall hepatic expression level
of certain ISGs and virus-responsive genes. This gene set was accurately defined as
those virus-responsive genes whose high expression associated with low viral load
and effective viral clearance but whose low expression correlated with progression to
chronic HCV infection (Su et al., 2002). Like many virus-responsive genes and ISGs,
the various products of the "outcome predictor" gene set are known to interact with
components of T cell immunity, again demonstrating the complex cross-talk that
goes on during the host response to infection and between parameters of innate and
adaptive immunity. These observations demonstrate that the hepatic host response
is triggered during HCV infection but differentially regulated in association with
disease course, and that HCV mediates persistence through strategies to regulate
and/or evade the antiviral actions of this response.

TRIGGERING THE HOST RESPONSE TO HCV INFECTION

The processes by which HCV initiates and controls the host response have been
addressed in cell culture models of HCV RNA replication and viral protein
expression. Genome-length or specific subgenomic fragments of HCV RNA are
sufficient to trigger IFN-β expression and production when introduced into cultured
human hepatoma cells (Fredericksen et al., 2001; McCormick et al., 2004), and
this effect has been attributed in part to the 5' and 3' nontranslated region (NTR)
of the HCV genome. These regions encode a series of highly conserved stem-loop/
dsRNA structures, and the 3' NTR includes a variable length poly u region (Tuplin

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et al., 2002). In cultured cells the HCV NTRs present PAMP structures that serve
as potent agonists of the host response. This suggests that during infection these
RNA motifs are recognized and engaged by PAMP receptor(s) that trigger the host
response (Sumpter et al., 2005).

The nature of at least one HCV RNA PAMP receptor was revealed through studies
of the Huh7-derived (human hepatoma) cell line, termed Huh7.5. This cell line
does not mount a host response to virus infection or transfected HCV RNA and
it is highly permissive for HCV RNA replication (Blight et al., 2002; Sumpter
et al., 2005). Complementation studies identified RIG-I as an HCV RNA PAMP
receptor that binds HCV dsRNA motifs and signals the activation of IRF-3 and
NF-κB. This was shown to induce IFN-β expression and onset of the host response
(Sumpter et al., 2005). RIG-I is an RNA helicase and a member of the DEx/D
box RNA helicase family. It contains amino-terminal regions of homology to the
caspase activation and recruitment domain (CARD) (Yoneyama et al., 2004). RIG-I
signaling is mediated by the CARD homology motifs while the helicase domain
imparts PAMP recognition, RNA-binding and regulation of signaling (Yoneyama
et al., 2004). Signaling by RIG-I directs a host response that suppresses HCV
RNA replication (Sumpter et al., 2005; Foy et al., 2005). The permissiveness of
Huh7.5 cells for HCV RNA replication has been attributed to a point mutation in
the RIG-I CARD motifs that ablated downstream IRF-3 phosphorylation and NF-
κB activation (Sumpter et al., 2005). Virus-induced signaling by RIG-I and the
resulting host response may therefore influence the outcome of HCV infection.
TLR3 is a dsRNA PAMP receptor that signals a host response after its engages the
PAMP ligand (Fig. 2) (Alexopoulou et al., 2001). TLR3 signals the activation of
IRF-3 and NF-κB through MyD88-independent processes that require the Toll-IL-1
receptor domain-containing adaptor inducing IFN-β (TRIF) protein (Yamamoto et
al., 2003). Genetic and biochemical studies have now defined prominent roles for
the RIG-I and TLR3 pathways in signaling the host response to virus infection in
hepatocytes (described below), though the role of each in natural HCV infection
remains to be evaluated.

Protein products of infection may also stimulate the host response to HCV.
Expression of the HCV NS5A protein induces cellular stress signaling pathways
to activate STAT3 (Gong et al., 2001). Similar to the IFN α/β receptor signaling,
STAT3 promotes gene expression through processes that involve the Jak-STAT
pathway (Sarcar et al., 2004). This results in a gene expression profile that includes
ISGs and proinflammatory cytokines that may influence the overall level of HCV
RNA replication (Zhu et al., 2003). Moreover, the HCV core protein has been
shown to activate the catalytic activity of protein kinase R (PKR), an ISG effector
component of the host response to virus infection (Delhem et al., 2001). PKR is
a dsRNA binding protein. Its activation is induced by binding to dsRNA PAMPs,

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resulting in eukaryotic initiation factor 2 phosphorylation and in inhibition of local

protein synthesis. Active PKR also signals the DNA binding activity of NF-κB and
induces IRF-1 transcription-effector action (Williams, 1999). Activation of PKR
by the HCV core protein is most likely attributed to the 5' NTR viral RNA binding
property of the core protein (Tanaka et al., 2000), which would provide the PKR
activator dsRNA PAMP required for kinase activation. Cell interaction with virus
particles may also trigger signaling events that induce IFN production. HCV pseudo
particle binding to dendritic cells has been shown to mediate particle uptake and
dendritic cell activation (Barth et al., 2005). Since dendritic cell subsets constitute a
major source of IFN production during viral infection, HCV modulation of dendritic
cell function may influence HCV infection by modulation local or systemic IFN
levels (Colonna et al., 2004).

REGULATION AND EVASION OF THE HOST RESPONSE BY

HCV
HCV utilizes a variety of strategies to regulate and evade the host response. Studies
of the HCV/host interface have revealed PAMP-responsive signaling pathways
that impart IRF-3 activation, IFN α/β receptor signaling, and ISG effector action
as major sites of control and evasion of the host response (Katze et al., 2002). The
HCV NS3/4A protease has been identified as an antagonist of virus-induced IRF-
3 activation and IFN-β expression. NS3/4A mediates a block to IRF-3 activation
triggered either by endogenous replicating HCV replicon RNA or by exogenous
virus infection of cells harboring a replicating HCV genomic RNA (Foy et al., 2003).
The IRF-3 blockade was been attributed to the NS3/4A protein complex, which
mediates a block to virus-induced IRF-3 phosphorylation, resulting in retention of
IRF-3 in an inactive, cytoplasmic-bound state. NS3 is a bifunctional enzyme, and
it encodes serine protease within its amino-terminal domain and a RNA helicase
within its carboxyl-terminal domain (Reed and Rice, 1998). Importantly, the NS3/4A
complex constitutes the essential viral protease, and it releases the nonstructural
proteins from the HCV polyprotein during virus replication (De Francesco and
Steinkuhler, 2000). The helicase activity of NS3 is not required for the control of
IRF-3 activation but the NS3/4A protease activity is required. This suggests that
HCV blocks IRF-3 activation through NS3/4A proteolysis of essential host cell
proteins that confer PAMP signaling (Foy et al., 2003). NS3/4A regulation of RIG-I
signaling has been identified as a causal link of the IRF-3 phosphorylation blockade
directed by NS3/4A. These studies showed that NS3/4A blocks the host RIG-I
pathway through the protease-dependent disruption of CARD-homology domain
signaling that is normally induced upon RIG-I binding to the HCV RNA PAMP
ligand. The NS3/4A block to RIG-I signaling additionally ablates virus activation
of NF-κB (Foy et al., 2005). This dual regulation of IRF-3 and NF-κB indicates
that NS3/4A must target and cleave common factor(s) involved in IRF-3 and NF-
κB activation but the nature of such factors have not been defined.

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TLR3 signaling is also targeted and regulated by NS3/4A. In this case NS3/4A
protease activity has been shown to cleave the TRIF adaptor protein between amino
acids C372 and S373 (Li et al., 2005b). This cleavage site is homologous with
the HCV NS5A/B polyprotein cleavage site. Structure/function studies show that
NS3/4A recognizes TRIF through binding of a proline-rich region adjacent to the
site of cleavage (Ferreon et al., 2005). TRIF is essential for signaling by TLR3 but it
does not play a role in RIG-I pathway, nor does TRIF cleavage by NS3/4A provide
a mechanism of RIG-I pathway regulation, which must occur through cleavage of
yet addition cellular targets (Foy et al., 2005; Li et al., 2005a). NS3/4A cleavage
of TRIF prevents TLR3 signaling, thus blocking IRF-3 and NF-κB activation and
preventing IFN production (Fig. 2) (Li et al., 2005b). The targeting of the RIG-I
and TLR3 pathways by NS3/4A allows HCV to evade two major arms of IFN
production and host defense. This has been further validated through pharmacologic
studies that used a peptidomimetic active site NS3 protease inhibitor to evaluate
the requirement for protease activity in the regulation of host response signaling by
NS3/4A. Treatment of cells that expressed wild type, functional NS3/4A showed
that the protease inhibitor effectively removed the blockade to RIG-I and TLR3
signaling imposed by HCV, thereby restoring virus-induced IRF-3 phosphorylation/
activation and the activation NF-κB (Foy et al., 2003; Foy et al., 2005). Protease
inhibitor treatment of cells also protected TRIF from cleavage by NS3/4A (Li et
al., 2005b).

What are the implications resulting from HCV disruption of RIG-I or TLR3
signaling? First, viral control of RIG-I and TLR3 serves to attenuate major
pathways of IFN production by infected cells and tissues. Second, many of the
components of these pathways are IFN-responsive and though expressed at low
levels, their expression is increased after exposure of cells and tissues to IFN α/β.
The IFN-responsiveness of these factors provides an amplification loop to enhance
the strength and length of the host response. The signaling blockade imposed by
NS3/4A breaks this IFN amplification loop (Foy et al., 2005) and may limit the
level and diversity of ISG expression induced in response to IFN therapy. Third,
HCV attenuation of the host response and IFN production is expected to cause
alterations in antigen presentation by the affected hepatic tissue, potentially leading
to inefficient activation of cytolytic T cells and an inability of the adaptive immune
response to clear HCV-infected hepatocytes by disrupting the cross talk between the
host response and the adaptive immune response. This may provide a causal link
between high level intrahepatic ISG expression and a vigorous T cell response to
diverse viral epitopes, both of which are associated with viral clearance and inversely
correlate with chronic infection (Su et al., 2002; Shoukry et al., 2004). Fourth,
IRF-3 has been ascribed proapoptotic and tumor suppressor functions (Heylbroeck
et al., 2000; Duguay et al., 2002). A prolonged block to IRF-3 activation might
disrupt these actions and cause a tumorigenic phenotype within infected cells. This

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could provide a link between chronic HCV and hepatocellular carcinoma (Liang
and Heller, 2004). Finally, the blockade of NF-κB function may interfere with a
variety of chemokines and cytokine genes whose expression is dependent on NF-
κB and serve to drive the inflammatory response to virus infection (Zhu and Liu,
2003; Foy et al., 2005). HCV regulation of NF-κB may therefore contribute to the
systemic immune defects associated with HCV infection.

HCV REGULATION OF IFN SIGNALING

The overall low response rate of HCV to IFN therapy, particularly among patents
with genotype 1 HCV infection (McHutchison et al., 2002), provides clinical
evidence that HCV can effectively evade IFN actions in vivo. Many studies have
focused on defining the molecular mechanisms by which HCV evades and resists
IFN actions. These studies have shown that HCV protein expression associates
with inhibition of STAT1 function, and can occur independently of STAT tyrosine
phosphorylation (Heim et al., 1999). Analysis of transgenic mice that express HCV
proteins in their hepatocytes showed that the regulation of STAT1 occurs below the
level STAT tyrosine phosphorylation, resulting in a defective hepatic IFN response
(Blindenbacher et al., 2003). STAT dysfunction might be attributed to protein
phosphatase 2A (Fig. 3), which confers STAT1 hypomethylation and complex
formation the protein inhibitor of activated STAT1 (PIAS). This was found to prevent
STAT1 assembly into the ISGF3 complex and to attenuate ISG expression (Duong
et al., 2004). The mechanism by which protein phosphatase 2A triggers these events
is not known. Others have shown that expression of the HCV core protein associates
with increased levels of suppressor of cytokine signaling (SOCS)-3 (Bode et al.,
2003). SOCS3 belongs to a family of SOCS proteins that are negative regulators
and inhibitors of Jak-STAT signaling. SOCS proteins mediate a classical negative
feedback loop on IFN α/β receptor signaling events; SOCS-1 and SOCS-3 confer
reduced levels of ISG expression (Alexander, 2002). While induction of SOCS-3
by the HCV core protein might impart evasion from IFN actions, it should be noted
that the overall role of SOCS-3 in HCV infection is not known and it is unclear
if expression of SOCS-3 by core is due to the potential for the core protein to
stimulate IFN signaling events itself (Miller et al., 2004) or through induction of
yet undefined signaling pathways that stimulate SOCS expression.

REGULATION OF ISG EXPRESSION OR FUNCTION

Molecular studies have linked HCV evasion of the host response and IFN therapy
with various strategies directed by viral proteins to control ISG expression or
function (Table 1). The HCV NS5A protein has been identified as an IFN antagonist.
Several studies have shown that expression of NS5A alone can attenuate IFN-α
actions and rescue the replication of IFN-sensitive viruses (Macdonald and Harris,
2004). Microarray analyses have shown that NS5A expression can confer a general
attenuation of ISG expression, though the mechanism of this regulation was not

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Fig. 3. HCV attenuation of IFN signaling. IFN α/β Receptor signaling by IFN from autocrine/paracrine
and therapeutic sources is subject to feedback inhibition by suppressor of cytokine signaling (SOCS)
proteins. The HCV core protein can induce the expression of SOCS-3, which suppresses Jak-STAT
signaling events (Alexander, 2002). Expression of the protein inhibitor of activated STAT (PIAS) is
induced by HCV proteins, possibly mediated by protein phosphatase 2A (PP2A) signaling events and
STAT demethylation (Duong et al., 2004). This blocks STAT1 function. Some patients with chronic
HCV infection exhibit aberrantly high levels of serum IL-8 (Polyak et al., 2001b). The biological
activity of IL-8 interferes with IFN signaling events (Khabar et al., 1997). HCV modulation of IFN
signaling allows the virus to evade the antiviral actions of the host response and IFN therapy.

defined (Geiss et al., 2003). NS5A induces interleukin (IL)-8 expression and
secretion. This has implications for IFN therapy because IL-8 is a proinflammatory
chemokine whose actions interfere with IFN (Fig. 3). The mechanism(s) of IL-8's
anti-IFN action may include attenuation of IFN signaling and ISG expression, and/or
direct inhibition of select ISGs (Khabar et al., 1997). Serum IL-8 levels are found
elevated in patients with chronic HCV, and NS5A has been shown to stimulate
IL-8 production through transactivation of the IL-8 promoter, possibly involving
NF-κB and AP-1 transcription factor activation by other cytokines (Polyak et al.,
2001a, Polyak et al., 2001b). IRFs may also drive IL8 production when activated
during virus infection (Casola et al., 2000). This latter point is another example
that serves to demonstrate the link between innate antiviral and pro-inflammatory
responses. In this case the link is exploited by NS5A as a means of evading the
host response to HCV infection.

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Table 1. Processes of ISG regulation or control by HCV.

Viral strategy Mechanism of Implications Reference/Example
action
IL-8 induction. NS5A induces IL8 Attenuates ISG Polyak et al., 2001a
production through expression.
processes involving
NF-κB and AP-1
transcription factor
activation.
Induction of SOCS The HCV core Blocks Jak-STAT Bode et al., 2003
expression. protein can induce signaling action through
expression of the IFN α/β receptor.
SOCS1 and SOCS3.
PKR inhibition NS5A and E2 Disruption of PKR- Taylor et al., 1999;
proteins bind PKR dependent translational Noguchi et al., 2001;
and inhibit its control and signaling Gimenez-Barcons et
catalytic activity. actions. al., 2005
IRF-1 regulation NS5A blocks ds Relieves IRF-1 Pflugheber et al.,
RNA induced IRF- suppression of HCV RNA 2002; Kanazawa et
1 action through replication. al., 2004
inhibition of PKR
signaling.
Evasion of 2'-5' OAS/ HCV genome The HCV genome Han et al., 2002; Han
RNase L pathway sequence. encodes a paucity of et al., 2004
RNase L recognition sites,
which allow protection
from nucleolytic
processing.
Disruption of STAT1 HCV proteins HCV proteins induce Heim et al., 1999;
function. PP2A expression and Duong et al., 2004
STAT1 hypomethylation
to attenuate ISG
expression.
Suppression of ISG56 HCV nonstructural In vitro: NS3/4A and Wang et al., 2002;
expression. proteins nonstructural proteins Sumpter et al., 2004
disrupt virus signaling
to the ISG56 promoter.
Removes the ISG56 block
to viral RNA translation.
Regulation of RIG-I NS3/4A protease Blockade of RIG-I Foy et al., 2005
signaling blockade of signaling breaks an IFN
signaling. amplification loop that
otherwise enhances ISG
expression.
Regulation of TLR3 NS3/4A protease Disruption of a Li et al., 2005b
signaling cleavage of TRIF TLR3-pathway IFN
amplification loop.
Activation of STAT3 The NS5A protein STAT3-target genes, Gong et al, 2001
induces a stress including some ISGs, are
response that expressed.
activates STAT3.

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The NS5A and E2 proteins of HCV have both been identified as inhibitors of
PKR (Gale, Jr. et al., 1998; Taylor et al., 1999; Noguchi et al., 2001). Inhibition of
PKR may allow HCV to evade in part the translational-suppressive and signaling
actions of PKR (Katze et al., 2002). This regulation is not universal and is subject
to alteration through viral genetic variation, such that not all NS5A sequences have
the ability to bind and inhibit PKR (Gimenez-Barcons et al., 2005). Thus, HCV
evasion of PKR-independent processes of the host response must also contribute to
HCV resistance to IFN. The product of the IFN-induced ISG56 gene (also known
as IFIT1 or the 561 gene), p56, can suppress HCV RNA translation (Wang et al.,
2002), and viral attenuation of ISG56 expression has been shown to associate
with a level of resistance to HCV RNA replication from the antiviral actions of
IFN in the HCV replicon cell culture model. ISG56 is both an ISG and an IRF-3-
target gene (Grandvaux et al., 2002), and the IFN-resistant phenotype in this case
associated with viral genetic adaptations that enhanced the NS3/4A blockade to
IRF-3 signaling (Sumpter et al., 2004). This raises the possibility that HCV control
of IRF-3 activation pathways may attenuate ISG expression by preventing the cross-
talk of cellular pathways that converge on ISG promoter elements. Studies that have
evaluated HCV interactions with the IFN-induced 2'-5' oligoadenylate synthetase
(OAS)/RNase L antiviral pathway have shown that HCV proteins interact with this
pathway (Taguchi et al., 2004). When activated, this pathway directs RNase L, an
endoribonuclease, to cleave the HCV genome RNA into nonfunctional products
(Han and Barton, 2002). RNase L cleaves HCV RNA only at certain UU and UA
dinucleotide sites (Han et al., 2004), and genotype 1 HCV sequences have overall
fewer RNase L cleavage sites than HCV genotypes 2 or 3 (Han et al., 2004). This
could provide a genetic mechanism for how, in part, HCV 1a and 1b infections resist
IFN therapy (Table 1). However, as described above and owing to the pleiotropic
nature of IFN effects mediated by the hundreds of ISGs, it is likely that HCV evades
IFN action through multiple strategies to redirect ISG functions.

VIRAL GENETICS IMPACT THE HOST RESPONSE TO HCV

INFECTION
The viral polymerase of HCV lacks proofreading function (Reed et al., 1998), and
during persistent infection the error-prone virus replication generates a repertoire
of highly related but genetically distinct viral variants or "quasispecies". This
provides a remarkable adaptive potential to HCV and has been implicated in
evasion and control of the host response and IFN therapy (Farci, 2001). A hostile
host environment may drive the outgrowth of HCV "evasion variants" from a pre-
existing quasispecies pool or through viral genetic adaptation. This idea is supported
by in vivo studies that evaluated viral sequences from the E1 and E2 coding regions
within patients with HCV infection. This work demonstrated viral genetic patterns
that associated with infection outcome and in which the resolution of acute HCV
infection consistently associated with an overall reduction in viral quasispecies

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complexity (Farci et al., 2000; Farci et al., 2002). In contrast, progression to chronic
infection and resistance to IFN therapy associated with increased viral genetic
complexity, suggesting that host immune pressure drives the outgrowth or selection
of viral evasion variants able to persist and resist IFN action. Analysis of the HCV
NS5A coding region has also identified specific domains that exhibit sequence
variation in patients with differential outcomes to IFN therapy. Meta-analyses
and long-term follow-up of these studies now provide support for NS5A sequence
variation within a 40 aa "interferon sensitivity determining region" (ISDR) that
associates with IFN therapy outcome (Enomoto et al., 1996, Pascu et al., 2004;
Schinkel et al., 2004). The ISDR is within a genetically-flexible domain that is a
major site of viral adaptations among HCV RNA replicons (Blight et al., 2000;
Appel et al., 2005). Thus, ISDR variation may affect the HCV replication fitness
and the host response to infection.

EXOGENOUS INDUCTION OF ANTIVIRAL HEPATIC DEFENSES

Various studies have described an absence or only a low level expression of IFN α/β
within liver tissue from patients with chronic HCV infection (Mihm et al., 2004).
This lack of high IFN α/β gene expression within the HCV-infected liver provides
indirect evidence that HCV imposes a blockade to IRF-3 activation in vivo and may
explain why some patients with chronic infection do not express significant levels
of hepatic ISGs. However, it fails to explain why other patients exhibit broad and
abundant ISG expression despite only low level IFN α/β expression in the infected
liver (Smith et al., 2003). The fact that hepatic ISG expression has associated with
the level and extent of liver pathology (Smith et al., 2003) suggests that ISGs might
be induced indirectly as a result of cellular stress from fibrosis and/or cirrhosis.
ISGs are also induced through STAT3 signaling; studies have shown that STAT3
is activated by NS5A, and that STAT3 activation occurs concomitantly with HCV
RNA replication (Gong et al., 2001; Waris et al., 2005). Viral activation of STAT3,
possibly mediated through stress-responsive signaling events, may contribute to
ISG expression during HCV infection (Fig. 4). ISGs may also be induced through
TLR engagement exogenously by extracellular products of damaged tissue or
viral replication. It is also noted that stress-induced cytokines, including TNF-α
and IL-1 can trigger IRF-1 expression and transactivation function, resulting in a
level of IFN-β production (Fujita et al., 1986). This could contribute to hepatic
ISG expression even when the RIG-I or TLR3 pathways are blocked by the HCV
NS3/4A protease. Exogenous immune effector cells that infiltrate the liver may
also contribute to hepatic ISG expression. In particular, IFN production by tissue
macrophages and dendritic cells that have infiltrated the infected tissue could lead
to a paracrine IFN response and ISG expression during the processes of antigen
cross-presentation in vivo (Schulz et al., 2005). By this model hepatic ISG levels
would vary with the composition and extent of immune cell infiltration, which has
been observed (Smith et al., 2003). Secretion of IFN-γ by T cells and NK cells

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Fig. 4. Processes of hepatic IFN production from exogenous sources. Various processes can result in
hepatic ISG expression despite a block to the host response imposed by the actions of HCV proteins.
In most of the examples shown, IFN will be produced from non-infected (exogenous) cell sources and
not from the chronic HCV-infected hepatocyte, which studies have shown do not express appreciable
levels of IFN (Mihm et al., 2004). NS5A triggering of STAT3 signaling (Waris et al., 2005) would
produce ISGs in the absence of IFN, and this affect would be limited to infected cells.

that have infiltrated the infected liver also contributes to ISG expression but the
pattern of expressed ISGs only partially overlaps with those induced by IFN α/β
(Der et al., 1998).

THE HCV/HOST INTERFACE MODEL OF VIRAL EVASION

Fig. 5 depicts a model of the virus/host interface and host response regulation
that forms a foundation for persistent HCV infection. The transmission event of
HCV infection presents unique pressures for the virus to adapt to the new host
environment. HCV adaptation to a new host will involve fine tuning of viral
strategies to control and evade the host response to infection. The transmission
event results in an acute infection that involves viral regulation of the host response
though RIG-I, TLR3 and other host defense signaling pathways within the infected
cell (Sumpter et al., 2005; Li et al., 2005a). Highly fit variants of HCV will mediate
signaling interference at the virus/host interface. This involves the actions of the
NS3/4A protease to block RIG-I and TLR3 signaling pathways. However, this
regulation is influenced by viral genetic variation, and genetic distinctions among the
many different quasispecies of HCV that are replicating in various cells at various
times post-infection will result in differential levels of control and activation of
this response. During acute infection the differential activation and control of the
host response by viral genetic variants will lead to the production of IFN and ISG
expression to mediate an antiviral state in the local hepatic tissue (Bigger et al.,
2001). 15-25% of all exposures to HCV typically result in acute/resolving infection
(McHutchison, 2004), indicating that a robust hepatic host response could provide
protection against the replication and spread of HCV. The host response and the
ensuing adaptive immune response present enormous pressures that will select
for the outgrowth of viral quasispecies that can evade and successfully control

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Fig. 5. HCV/host interactions regulate infection outcome. The model is described in the text, and its
shows a flow of events in which the virus/host interface and interactions within the host response will
decide the fate of HCV infection. By this model, HCV disruption of the host response to infection
provides a the frame work for viral persistence and chronic infection, may contribute resistance to
IFN therapy.

the host response and immune defenses (Farci et al., 2000; Sumpter et al., 2004).
HCV/host interactions at key sites of host defense signaling serve to suppress the
host response, attenuate the actions of IFN therapy, and provide a solid foundation
for persistent HCV infection. This model incorporates the important, variable
and perhaps unpredictable aspect of viral adaptation and quasispecies selection
within the evasion and control strategies by which HCV limits the host response
to infection. Further studies to identify the viral genetic elements (including viral
PAMPs and PAMP structure), signaling factors and ISG effectors that regulate the
host response to infection will certainly increase our understanding of host defense
and the HCV/host interface that controls infection outcome.

ACKNOWLEDGEMENTS
The Gale laboratory is supported by grants from the NIH, the Ellison Medical
Foundation and the Burroughs Wellcome Fund. DS is supported by NIH training
grant 5T32DK007745. M.G. is the Nancy C. and Jeffrey A. Marcus Scholar in
Medical Research in Honor of Dr. Bill. S. Vowell.

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NOTE ADDED IN PROOF

Recent studies have shown that NS3/4A cleaves the host Cardif/iPS-1 protein
to block RIG-1 signaling during infection. Meylan, E., Curran, J., Hofmann, K.,
Moradpour, D., Binder, M., Bartenschlager, R., and Tschopp, J. Cardif is an adaptor
protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature
437, 1167-1172.

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Chapter 14

Regulation of Adaptive Immunity by HCV

Xiao-Song He

ABSTRACT
HCV causes chronic infection in the majority of infected patients, which is
associated with attenuated adaptive immunity against the virus. Accumulating
data suggest that HCV may modulate the adaptive anti-HCV immunity of the
host to facilitate the establishment of viral persistence. Potential mechanisms of
this modulation include infection of dendritic cells by HCV, as well as binding of
HCV envelope or core proteins to cell surface receptors, resulting in perturbation
of the functions of different immune cell subsets. These mechanisms may operate
predominantly in the liver, the primary site of infection by HCV, where the unique
hepatic environment favors tolerance rather than immunity to foreign antigens.
Elucidation of these mechanisms may lead to development of novel therapeutic
strategies combining both antiviral drugs and immunotherapy agents.

INTRODUCTION
Hepatitis C virus (HCV) is a major blood-borne virus that infects over 100 million
people worldwide and 2.7 million in the United States. It is estimated that in less
than 20% of HCV-infected individuals the virus is cleared spontaneously, while in
the majority of patients the virus persists and causes chronic hepatitis that may lead
to end-stage liver diseases requiring liver transplantation (Alter et al., 1999). The
mechanisms underlying different outcomes of infection are not clear at this time.
The host immune responses, including innate immunity and adaptive immunity, play
a critical role in determining the outcome of viral infection, as well as in the nature
and extent of liver cell injury during HCV infection (Rehermann and Chisari, 2000;
He and Greenberg, 2002). Since the rate of persistence for HCV is much higher
than other hepatitis viruses, for example, hepatitis B virus (HBV) that persists in
only less than 10% of immunocompetent adults who are infected (Hollinger and
Liang, 2001), HCV appears to be more successful than many other viruses in terms
of evading the protective immunity of the host. However, little is known regarding
the exact reasons for the failure of the host immune system in fighting HCV. In
this article the current knowledge regarding the adaptive immunity to HCV will
be reviewed first, followed by a discussion on the potential mechanisms HCV may
employ to interfere with the normal functions of host immune system to achieve
its persistence.

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ADAPTIVE IMMUNITY TO HCV: A FAILURE IN MOST PATIENTS

Although HCV persists in the majority of infected individuals, a small fraction
of patients can successfully clear the infecting virus. In a study on injection drug
users, those who resolved previous HCV infection were 12 times less likely to be
reinfected to develop persistent infection than people infected for the first time.
In those who did become reinfected, the median peak HCV RNA levels were two
logs lower than people infected for the first time to develop persistent infection.
These findings suggest that a protective immunity does exist, which is capable of
complete or partial control of HCV infection (Mehta et al., 2002).

While the role of neutralizing antibodies in the protective immunity against HCV
has recently regained attention (Hsu et al., 2003; Logvinoff et al., 2004), most
of the previous studies on the human adaptive immune responses against HCV
focused on the T cell responses. Although the number of cases with self-limited
HCV infection that have been carefully studied is relatively small, such studies
usually reveal a vigorous HCV-specific T cell response, including CD4 T cell
response (Gerlach et al., 1999; Takaki et al., 2000; Thimme et al., 2001; Rosen
et al., 2002) and CD8 T cell response (Gruner et al., 2000; Lechner et al., 2000b;
Takaki et al., 2000; Thimme et al., 2001; Lauer et al., 2004). These responses were
detected early during the acute phase (Takaki et al., 2000; Thimme et al., 2001) and
sustained for many years after the clearance of HCV (Takaki et al., 2000). They
were usually broadly targeted at multiple epitopes restricted by different MHC
molecules, without a dominant epitope (Cooper et al., 1999; Lauer et al., 2004).
In contrast, patients with chronic HCV infection usually have weak or defected T
cell responses against HCV, as indicated by low frequencies for the specific T cells
(He et al., 1999; Lauer et al., 2004), short-lived responses (Lechner et al., 2000a;
Ulsenheimer et al., 2003), narrowly targeted epitopes (Lauer et al., 2004), as well
as defects in the effector functions of the specific T cells (Gruener et al., 2001;
Wedemeyer et al., 2002). Taken together, these studies strongly suggest that the
host T cell responses are a key factor in determining the outcome of HCV infection.
Of note, during the acute phase of self-limited HCV infection, a brief period of
dysfunction of HCV-specific CD8 T cells has also been documented (Lechner et
al., 2000b; Thimme et al., 2001), suggesting that a transient down-modulation of
the effector functions of specific CD8 T cells may be a host strategy to limit the
tissue damage caused by the cytotoxic CD8 T cells at the early stage of infection
when viral replication is at its peak rate.

Although it is still unclear why T cell responses fail to clear HCV in most cases,
a comparison of T cell immunity against HCV and other viruses with different
outcomes of infection has generated some intriguing results. Within the category of
persistent viruses, the pattern of viral replication varies from latent infections that
undergo periodic reactivation (e.g. Epstein-Barr virus, EBV), to ongoing low-level

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viral replication (e.g. human cytomegalovirus, CMV) controlled by the immune

responses without causing disease, to persistent high-level viral replication subjected
to immune control to variable extents at different stages of disease course, as in
the case of HIV. MHC class I tetramers and cytokine flow cytometry assays have
been used to characterize phenotypes of human CD8 T cell responses to persistent
viral pathogens (He et al., 1999; Appay et al., 2000; Gamadia et al., 2001; Hislop
et al., 2001; Catalina et al., 2002; Khan et al., 2002; Wedemeyer et al., 2002).
A study comparing the phenotypes of peripheral CD8 T cells specific for HIV,
CMV, EBV and HCV showed that the expression of CD8 T cell surface markers
thought to be related to CD8 T cell differentiation, including CD27 and CD28,
were heterogeneous between CD8 T cells specific for different viruses (Appay et
al., 2002). The authors proposed the use of CD27 and CD28 expression to classify
virus-specific CD8 T cells into early (CD27+CD28+), intermediate (CD27+CD28–)
and late (CD27–CD28–) differentiated cells. Interestingly, CD8 T cells specific
for each of the four viruses appeared to fall into different stages based upon this
classification; that is, HCV-specific CD8 T cells had markers associated with the
early differentiation phenotype, EBV-specific CD8 T cells were classified as early-
intermediate, CD8 T cells recognizing HIV antigens were intermediate, and only
CMV-specific CD8 T cells had the proposed late phenotype. Of note, memory CD8
T cells specific for influenza A virus (fluA), which causes transient infections and
is cleared by the immune system, have phenotypes consistent with those of early
differentiated cells (He et al., 2003).

Although the mechanisms explaining these heterogeneous phenotypes of virus-

specific T cells are not known, there appears to be a correlation between the
differentiation stage of virus-specific CD8 T cells and the control of viral replication
mediated by the CD8 T cell response. CMV is a persisting virus with ongoing
low-level viral replication, but it does not cause any disease in immune competent
subjects; this is associated with high levels of fully differentiated effector CD8 T
cells that control the ongoing replication of virus. On the other end of the spectrum,
fluA does not persist in infected host. The fluA-specific memory T cells responsible
for the long-term maintenance of immunity in healthy subjects are experiencing rest
from stimulation by viral antigens (Wherry and Ahmed, 2004); this is associated
with their early differentiation phenotypes. Between these two extremes lies EBV,
which is a latent virus with periodic reactivation that re-stimulates the resting
memory T cells briefly at intervals, resulting in an early-intermediate phenotype of
the EBV-specific CD8 T cells. HIV causes a persistent and progressive infection.
This is associated with an intermediate differentiation phenotype of HIV-specific
CD8 T cells with certain functional defects when compared to CMV-specific CD8
T cells (Appay et al., 2000; Champagne et al., 2001). Such an immune response
may partially control the ongoing viral replication during the asymptomatic phase,
until the depletion of helper CD4 T cells leads to the collapse of the immune system
and onset of AIDS.

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However, when HCV-specific CD8 T cells are compared to CD8 T cells specific
for the other viruses, they do not fit into the general pattern described above. While
HCV causes persistent infection and ongoing disease, HCV-specific CD8 T cells,
if ever detected in the peripheral blood, are frequently found to have a phenotype
consistent with an early differentiation stage, similar to the resting fluA-specific
memory CD8 T cells which had not been exposed to the virus since the last acute
influenza was cleared. Some of the HCV-specific CD8 T cells even appeared to
be functional when stimulated ex vivo with the endogenous viral peptides of the
patient (He et al., unpublished data). In other words, HCV-specific CD8 T cells in
many patients appear to be in a state of rest or anergy in vivo, ignoring the ongoing
HCV infection. Of particular interest is a recent study on a cohort of patients
co-infected with HCV and CMV, which found that CMV-specific CD8 T cells in
these patients appeared to have lost some markers associated with differentiation
maturity, including increased expression of CCR7 and reduced expression of Fas
and perforin, although they maintained functional responses to in vitro stimulation
with CMV antigen (Lucas et al., 2004). The authors suggested that the reduction in
mature CD8 T cells in HCV-infected individuals arises through either impairment
or regulation of T cell stimulation, or through the early loss of mature T cells. In
either case, HCV may have a pervasive influence on the general T cell immunity
of infected hosts, which is not limited to HCV-specific T cells.

A critical factor for the development and differentiation of T cells into functional
memory and effector cells is the stimulation that they receive during the primary
response (Lanzavecchia and Sallusto, 2002). In order to understand the apparent
weak or abnormal T cell immunity to HCV, it is important to investigate the initial
events leading to the antiviral adaptive immunity, which is the processing and
presentation of viral antigens.

DENDRITIC CELLS – ARE THEY INFECTED BY HCV?

While B and T lymphocytes are the effectors of the adaptive immunity, their
development and function is under the control of dendritic cells (DCs). Different
subsets of DCs, including myeloid-derived DCs (MDC) and plasmacytoid-derived
DCs (PDC), are the most important antigen presenting cells with the capability
to capture and process antigens, express lymphocyte co-stimulatory molecules,
migrate to lymphoid organs and secrete cytokines to initiate B and T cell responses
(Banchereau et al., 2000). DCs not only activate lymphocytes, they also have the
important function of tolerizing T cells to self-antigens, which results in the anergic
state of such cells to avoid autoimmune reactions (Banchereau et al., 2000). The
two opposite roles of DCs have been related to different maturity and cytokine
production patterns of DCs (Crispe, 2003; Kubo et al., 2004). Thus, depending on
the nature of antigens as foreign or self, the functions of DCs have to be precisely
controlled to either activate or tolerize T cells. Any perturbation to this control

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may lead to serious consequences. These include impaired immunity to infecting

pathogens, causing persistent infections; or abnormal immunity to self-antigens,
causing autoimmune diseases. In addition to functioning as antigen presenting cells
that play a key role in the induction of adaptive immunity or immune tolerance, DC
is also an important part of the antiviral innate immunity which is largely mediated
by the type I interferons (IFNs) produced by PDCs (Cella et al., 1999).

Many viruses can directly infect DCs through different cell surface receptors. In
response to this invasion, DCs process viral proteins and present them through
MHC class I and II pathways while undergoing a maturation that enhances their
presentation of antigen to T cells and expression of T cell costimulation factors
for induction of adaptive T cell antiviral immunity (Carbone and Heath, 2003;
Rinaldo and Piazza, 2004). As a strategy to counteract antiviral immunity, some
viruses have evolved mechanisms to undermine the functions of DCs. For example,
infection of DCs by measles virus resulted in diminished IL-12 production and
inhibition of DC maturation (Schneider-Schaulies et al., 2003; Servet-Delprat et
al., 2003). HIV-infected subjects had defects in the number, immunophenotype and
functions of blood DC subsets infected with HIV (Barron et al., 2003; Donaghy et
al., 2003). Infection of DCs by CMV has also been shown to cause inhibition of
DC maturation and T cell activation, as well as increased production of molecules
that induce apoptosis in T cells and down-regulation of MHC class I molecules
(Raftery et al., 2001; Moutaftsi et al., 2002).

Accumulating evidence suggests that DCs are susceptible to HCV infection. In

some studies, HCV RNA sequences, including replicative negative-strand RNA,
have been detected in DCs isolated from patients with chronic HCV infection
(Bain et al., 2001; Goutagny et al., 2003; Tsubouchi et al., 2004a). It was reported
that both immature and mature monocyte-derived DCs were infected with HCV,
as indicated by detection of negative-strand HCV RNA in the cells incubated in
vitro with HCV-positive serum samples (Navas et al., 2002). In a study of HCV
RNA sequencies isolated from liver and DC samples of a patient, the quasispecies
detected in DCs were unique and differed from those present in the liver, suggesting
a particular tropism of HCV quasispecies for DCs. Moreover, the translational
activity of DC-derived HCV was significantly impaired when compared with those
from liver and PBMCs, suggesting an impaired replication of HCV in the DCs
(Laporte et al., 2003). Infection of DCs by HCV is thought to be mediated by the
interaction of the HCV glycoprotein E2 with DC-SIGN, a membrane-associated
C-type lectin that also involves in the binding of HIV to DCs (Lozach et al., 2003;
Pohlmann et al., 2003).

Several groups have reported dysfunction of DCs that may potentially affect
adaptive immunity in patients with persistent HCV infection, using various in vitro

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functional assays for DCs. These include impaired allostimulatory abilities to CD4
T cells (Kanto et al., 1999; Auffermann-Gretzinger et al., 2001; Bain et al., 2001;
Kanto et al., 2004; Tsubouchi et al., 2004a), defects in responding to maturation
stimuli (Auffermann-Gretzinger et al., 2001), as well as impaired ability to secrete
IL-12, a cytokine important for the development of CD4 helper T cell responses
(Anthony et al., 2004; Kanto et al., 2004). Some of these defects were reversed after
IFN-α therapy that cleared HCV in the sera (Auffermann-Gretzinger et al., 2001)
or DCs (Tsubouchi et al., 2004b), indicating that the DC dysfunction is associated
with HCV infection. Interestingly, a positive association was observed between
MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-
infected subjects (Anthony et al., 2004). The DC-mediated innate antiviral immunity
also appeared to be impaired in HCV-infected patients, as indicated by reduced
production of IFN-α by PDCs (Anthony et al., 2004; Kanto et al., 2004). Of note,
IFN-α is not only an important antiviral cytokine; it is also an important modulator
for adaptive immunity. It has been reported that IFN-α enhances expression of class
I and class II molecules, cytokines and perforin that are involved in the presentation
of viral antigens and the effector functions of T cells (Ji et al., 2003), as well as
provides a stimulating signal for the clonal expansion and differentiation of CD8
T cells (Curtsinger et al., 2005).

In addition to the dysfunction of DCs, the numbers of MDCs, PDCs and DC

progenitors in the periphery were significantly lower in patients with chronic
hepatitis than in healthy controls (Kanto et al., 2004; Wertheimer et al., 2004).
Taken together, these findings point to a defective immune response mediated by
HCV-induced DC dysfunction as a potential mechanism enabling the persistent
HCV infection.

It should be mentioned that most of the reported DC dysfunctions were based

on monocyte-derived DCs generated in vitro. While this system has been used
extensively in studying DC biology, it is not clear to what extend this model
represents the functions of DCs in vivo (Kanto and Hayashi, 2004), especially those
in the infected liver. In addition, conflicting results have been reported by other
investigators who had conducted similar studies but failed to detect monocyte-
derived DC dysfunction in HCV chronically infected patients (Longman et al.,
2004) or chimpanzees (Rollier et al., 2003; Larsson et al., 2004), the only animal
model for HCV infection. Obviously, more studies are warranted to elucidate the
exact role of DCs in the apparent HCV-specific immunodeficiency.

INTERFERENCE OF HOST CELL FUNCTIONS BY HCV

While it is still controversial in terms of DC dysfunction in HCV-infected patients,
in vitro studies have shown that various HCV proteins have the potential capability
to modulate host cell functions by interfering with cellular signal transduction.

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Numerous interactions between HCV proteins and cellular components have been
identified in cell lines or experimental mice by using different expression systems
for HCV proteins (Tellinghuisen and Rice, 2002). Studies have showed that the
expression of HCV proteins suppressed IFN-induced signal transduction through
the JAK-STAT pathway (Heim et al., 1999; Blindenbacher et al., 2003; Geiss et
al., 2003; Duong et al., 2004). Specifically, a recent study in transfected cell line
demonstrate that expression of HCV proteins suppressed IFN signaling by degrading
STAT1, a major signal protein of the JAK-STAT pathway (Lin et al., 2005). HCV
NS3/4A serine protease blocks viral activation of IFN regulatory factor-3 (IRF-3),
a key transcription factor in inducing type I IFN expression, by proteolytic cleavage
of a cellular protein in the IRF-3 signaling pathway (Foy et al., 2003), while HCV
NS5A and E2 inhibits the IFN-inducible protein kinase PKR thought to play a role
in the antiviral effect of IFN (Gale et al., 1999; Taylor et al., 1999). In addition to
their central role in the innate immunity against viral infections, type I IFNs also
exert modulation functions to the adaptive antiviral immunity (Boehm et al., 1997;
Foster, 1997; Ji et al., 2003; Diepolder, 2004; Curtsinger et al., 2005). Of particular
interest, DCs are a key component of both innate immunity and adaptive immunity,
which orchestrate a successful overall immune response against infecting viruses.
In an intriguing in vivo mouse study, Sarobe et al. used recombinant adenovirus
vectors encoding HCV core/E1 or NS3 proteins to demonstrate that expression of
specific HCV proteins in DCs down-modulated the antiviral adaptive immunity
(Sarobe et al., 2003). The authors found that the expression of core/E1 proteins
in DCs inhibited their maturation. When mice were immunized with immature
DCs transduced with an adenovirus encoding core/E1, lower CD4 and CD8 T cell
responses were induced in comparison to the mice receiving DCs transduced with
an adenovirus encoding NS3.

In addition to the effects of intracellularly expressed HCV proteins, the interference

of cellular functions by extracellular HCV proteins binding to cell surface receptors
has also been documented. CD81 is a cell surface marker that binds the major
envelope protein E2 of HCV (Pileri et al., 1998), and has been suggested to be
involved in the infection process of HCV (McKeating et al., 2004; Zhang et al.,
2004). It was reported that engagement of CD81 with exogenous HCV E2 affected
multiple functions of natural killer (NK) cells including activation, cytokine
production, proliferation, and cytotoxic granule release (Crotta et al., 2002; Tseng
and Klimpel, 2002). While the NK cell is a primary effector of the innate immune
system, a complex interaction exists between NK cells and DCs (Andrews et al.,
2005), which may lead to activation or killing of DCs, depending on the nature of
the interaction (Ferlazzo et al., 2002; Gerosa et al., 2002; Moretta, 2002; Piccioli
et al., 2002; Zitvogel, 2002). This indicates an important role of NK cells in the
regulation of adaptive immunity to infections. It has been shown in a mouse model
that NK cells are necessary for optimal priming of adenovirus-specific T cells (Liu

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et al., 2000). Of particular interest, in a study on the regulation of NK cell activities

by inhibitory receptor CD94/NKG2A that normally leads to NK cell-induced
activation of DCs, NK cells from chronic HCV-infected donors were not capable
of activating DCs under the same conditions. In comparison to NK cells from
normal donors, those from HCV-infected patients showed higher expression of the
inhibitory receptor CD94/NKG2A and the cytokines IL-10 and TGF-β (Jinushi et
al., 2004). Therefore, modulation of NK cells could be another potential pathway
for HCV to affect the host innate and adaptive immune responses.

While E2 is a major component of HCV envelope and is readily available in HCV-

positive serum for interaction with cell surface receptors, it was reported that free
HCV core protein was secreted from stable transfectant cell lines (Sabile et al., 1999)
and could be detected in the serum of HCV-infected patients as well (Maillard et
al., 2001). In a series of publications, Hahn and colleagues reported in vivo and in
vitro studies suggesting that HCV core acts as an immunomodulator for the host
T cell response. They first demonstrated that the core protein of HCV genotype
1a delivered by a recombinant vaccinia vector suppressed the immune response
to the vaccinia virus in mice (Large et al., 1999). Using in vitro cell systems, they
further showed that the core protein bound to the complement receptor gC1qR
on T cells and inhibited their proliferation and IFN-γ production (Kittlesen et al.,
2000; Yao et al., 2001; Yao et al., 2003; Yao et al., 2004). Based on these results, a
model has been proposed that HCV core acts as an immune modulator that binds
to a component of the host complement system and suppresses T cell responses,
leading to the persistence of HCV (Eisen-Vandervelde et al., 2004). This is an
attractive model because the binding of C1q, the natural ligand for gC1qR, to T cells
is already known to suppress T cell response (Chen et al., 1994). In addition, other
pathogens, including measles virus, EBV, and HIV, also appear to exploit similar
strategies to suppress the host immune system by interactions with components of
the host complement machinery (Fingeroth et al., 1984; Viscidi et al., 1989; Karp
et al., 1996).

However, the role of HCV core as an immunomodulator is still an issue of debate,

as similar studies using the core of HCV genotype 1b delivered with a recombinant
adenovirus vector, or using genotype 1b core transgenic mouse, both failed to
demonstrate any immunomodulatory effects on virus-induced cellular immunity
(Sun et al., 2001; Liu et al., 2002). If the immunomodulator function of HCV
core is a unique feature of HCV genotype 1a but not genotype 1b, this does not
explain the fact that both HCV strains are equally likely to establish persistent
infection. Therefore, this model needs direct evidence from human clinical studies,
as well as more vigorous testing with in vivo experimental systems, including
chimpanzees.

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 Does HCV Cause Dysfunction in the Immune Cells?

REGULATORY T CELLS IN HCV INFECTION

Regulatory T cells (Tregs) have been recognized to be an important modulator
of T cell immunity in recent years. The most studied Tregs are those with the
phenotype CD4+CD25+, which have been shown to be powerful inhibitors of T
cell activation both in vivo and in vitro (Shevach, 2002). While the involvement
of Tregs in human autoimmune diseases such as multiple sclerosis and myasthenia
gravis has been established (Viglietta et al., 2004; Balandina et al., 2005), the
potential role of Tregs in viral hepatitis is just beginning to be defined. They could
limit liver injury by controlling inflammation, or they may promote persistence of
infection by suppressing immune responses (Chang, 2005). Recently, increased
numbers of these cells have been linked to the impaired immune response in patients
with chronic HBV infection (Stoop et al., 2005). In chronic HCV infection, the
persistence of HCV was associated with a reversible CD4-mediated suppression
of HCV-specific CD8 T cells and with higher frequencies of CD4+CD25+ Tregs
that could directly suppress HCV-specific CD8 T cells ex vivo (Sugimoto et al.,
2003; Cabrera et al., 2004). However, these studies did not answer the question of
whether the abnormalities of Tregs associated with persistent HCV infection are
the causes or the consequences of chronic HCV infection.

Studies in mice have linked the immune regulatory function of Tregs to DCs (Pasare
and Medzhitov, 2003). It was reported recently that the suppressive function of
Tregs was critically dependent on immature DCs and was readily reversed by the
maturation of DCs (Kubo et al., 2004), indicating that the maturity of DCs is a
key factor that determines suppression or activation of adaptive immune response.
Therefore, if the functions of DCs are indeed modulated by HCV infection, Tregs
may provide another potential pathway for HCV to manipulate host adaptive
immunity to benefit its persistence.

IMMUNE CELLS AND IMMUNE RESPONSES IN THE LIVER

Fig. 1 is a summary of the aforementioned potential mechanisms for the regulation
of adaptive immunity by HCV. It should be emphasized that these models are largely
based on in vitro or ex vivo experiments using human immune cells isolated from
peripheral blood or on mouse experiments and have not been verified in HCV
infected patients. A major challenge to all these potential mechanisms, or the HCV-
induced dysfunctions of different immune cell subsets including DCs, NKs and T
cells, is that they are not in agreement with the lack of global immune deficiency
in HCV-infected individuals similar to that in HIV-infected individuals. In other
words, these mechanisms cannot explain why only HCV-specific immune responses
are impaired, while the immune responses against other pathogens appear to be
spared from the HCV-mediated immune dysfunctions. Although a recent study
suggested that in patients with chronic HCV infection the phenotypes of CMV-
specific CD8 T cells were affected, there was no evidence for functional defects
of CMV immunity (Lucas et al., 2004).

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Fig. 1. Potential mechanisms for HCV-mediated interference of adaptive immunity. All these
mechanisms are likely to operate primarily in the liver.

The primary site of HCV replication and the major location of disease caused
by HCV are both in the liver, where most of the immunopathologic events
associated with the infection are likely to occur. This notion is supported by the
dramatic lymphocyte infiltration in the inflamed liver, but not in the normal liver.
Unfortunately, because of the difficulty in obtaining liver specimens, most of the
immunological studies on human liver diseases have to rely on peripheral blood
samples. Although very little is known about the immune cells in human liver
compared to their counterparts in the peripheral blood, evidences derived from
limited studies that directly investigated immune cells in normal and HCV-infected
livers have revealed significant differences between the intrahepatic and peripheral
lymphocyte subsets, including their activation status, phenotypes and proliferation
capability (Nuti et al., 1998; Wang et al., 2004; Ward et al., 2004). By using MHC
tetramers, HCV-specific CD8 T cells in the liver have been characterized directly.
These studies revealed that such cells were enriched in HCV-infected liver versus
peripheral blood and had different surface phenotypes compared to their counterparts
in the periphery (He et al., 1999; Grabowska et al., 2001; Accapezzato et al., 2004).
Of note, studies in mice have demonstrated a highly heterogeneous nature of hepatic
DCs and identified unique intrahepatic DC subsets with phenotypic and functional
features distinct from DC subsets isolated from other sites (Lian et al., 2003).

408
 Does HCV Cause Dysfunction in the Immune Cells?

As the largest organ in the body, the liver not only has various excretory, detoxifying
and metabolic functions, but it is also considered an intrinsic lymphoid organ
(Mackay, 2002) with unique microenvironment compared to other lymphoid
tissues in the periphery, such as the lymph nodes. Therefore, naive T cells in the
liver may encounter local antigens and start development and differentiation in a
manner distinct from those in the periphery, including such dramatic differences
as apoptosis versus proliferation (Park et al., 2002). It has been shown in mice that
oral administration of a foreign antigen at high dosage generated CD4 Tregs that
suppressed T cell proliferation as well as Ab responses to the antigen (Watanabe et
al., 2002). Of particular interest, Bowen et al. recently demonstrated in mice that the
site of primary T cell activation is a determinant of the balance between intrahepatic
tolerance and immunity. They showed that while naive CD8 T cells activated within
the lymph nodes were capable of mediating hepatitis, cells undergoing primary
activation within the liver exhibited defective cytotoxic function and shortened
half-life and did not mediate hepatocellular injury (Bowen et al., 2004). These
findings emphasized the unique nature of immune responses in the liver versus
the periphery.

The intrahepatic tolerance can be considered a requirement for the special functions
of the liver. The incoming blood stream from the intestine to the liver carries large
amount of food-derived antigens that are foreign in nature but mainly harmless
to the body. The constant presence of non-self antigens in the liver is thought to
impose a constraint on the immune responses generated in the liver, resulting in
a tolerant environment for foreign antigens (Crispe, 2003). The unique tolerance
nature of liver is best demonstrated by the fact that allogeneic liver transplantation
can be established without immunosuppression (Calne et al., 1969). However, this
constraint on liver immunity does not prevent the immune system from mounting
vigorous responses against some liver-specific pathogens such as hepatitis A virus,
which is almost always cleared after a self-limited infection (Hollinger and Emerson,
2001), and HBV, which is also cleared in more than 90% of immunocompetent
adults (Hollinger and Liang, 2001). Obviously, a precise control on the actions of
the intrahepatic immune cells is in operation, leading to either tolerance or immunity
to foreign antigens.

Although the controlling mechanism for liver tolerance is poorly understood at

this time, it is reasonable to speculate that liver resident antigen presenting cells,
including DCs, liver sinusoidal endothelial cells (Knolle and Limmer, 2001) and
liver resident machrophages or Kupffer cells (Everett et al., 2003), play a critical
role in shaping the outcome of intrahepatic immune responses. In addition to
the professional antigen presenting cells, hepatocytes may also serve as antigen
presenting cells under certain conditions (Herkel et al., 2003). It has been shown in
mice that liver sinusoidal endothelial cells selectively suppressed the expansion of

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IFN-γ-producing Th1 cells but promoted the outgrowth of IL-4-expressing Th2 cells,
creating an immune suppressive milieu that favors development of tolerance rather
than immunity within the liver (Klugewitz et al., 2002). In particular, the accessory
signals delivered by the hepatic antigen presenting cells, including cytokines and
costimulating molecules, are likely to exert profound effects on the regulation of
intrahepatic T cell immunity (Crispe, 2003). Given that liver is the primary site
of replication for HCV with the highest concentration of viral protein products, it
is conceivable that HCV-mediated interference of immune cell functions (Fig. 1),
including those of HCV-infected DCs or other antigen presenting cells as well as
NK cells and T cells, occurs primarily in the liver rather than other sites of the body,
resulting in a suppressed immunity against HCV but relatively unaffected immune
responses against other pathogens that do not primarily infect the liver. Future
studies on the issue of HCV-mediated immune modulation should focus on the
relevant events in the liver that affect the function of intrahepatic immune cells.

CONCLUSION
Although the mechanism for HCV to evade host immune responses and establish
chronic infection is still poorly understood, accumulating data indicate that HCV may
play an active role in attenuating host adaptive immunity to benefit its persistence.
Therefore, suppression of HCV replication by antiviral treatment should restore the
T cell immunity against HCV. Indeed, in HCV chronically-infected patients treated
with IFN, increased T cell immunity after IFN therapy has been demonstrated for
HCV-specific CD4 T cells (Cramp et al., 2000; Barnes et al., 2002; Kamal et al.,
2002) and CD8 T cells (Vertuani et al., 2002; Morishima et al., 2003), although in
some studies an increase in HCV-specific CD8 T cells was not detected (Barnes
et al., 2002). The discrepancy could be caused by different methods and antigens
used to measure CD8 response and should be resolved by further studies.

While current pegylated IFN-based anti-HCV therapies have accomplished greatly

improved efficacy, the rate of sustained virological response is still less than 50%
for the most common genotype of HCV (Manns et al., 2001; Fried et al., 2002). In
a significant fraction of patients who failed to achieve long-term response, however,
the HCV replication was temporally suppressed during IFN treatment. This may
represent an opportunity for immunotherapy such as therapeutic vaccines designed
to enhance adaptive immunity against HCV. With the HCV viral load suppressed
by IFN, the therapeutic vaccine is likely to elicit an anti-HCV immunity most
efficiently. Therefore a combination of antiviral drugs and a therapeutic vaccine
may produce a synergetic effect that surpasses the potential of either treatment
strategy alone.

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 Does HCV Cause Dysfunction in the Immune Cells?

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Chapter 15

Recombinant Vesicular Stomatitis Virus

(VSV) and Other Strategies in HCV
Vaccine Designs and Immunotherapy
Ayaz M. Majid and Glen N. Barber

ABSTRACT
Several vaccine strategies have been attempted in chimpanzee and smaller animal
models to generate immune responses to hepatitis C virus (HCV). While neutralizing
antibody may play a role in preventing HCV infection, studies in chimpanzees and
humans during rare cases of acute resolving HCV infection indicate that, HCV
immunity appears to be associated with vigorous, sustained and multi-specific Th1
intra hepatic CD8+ and CD4+ T cell responses. Several new promising technologies
utilizing viral based vaccine approaches that appear to generate both antibody and
cell mediated immune responses have recently been reported. These include viral
vectors that express HCV products and non-replicating viral like particles (VLPs)
that appear to induce T-helper type 1 (Th1) immune responses considered important
in resolving HCV infection. In addition, viral vectors based on recombinant vesicular
stomatitis virus (rVSV) may offer safe yet potent stimulation of both innate and
adaptive immune responses. Here, we review the successful application of viral
based vaccines, including VSV in generating viral immunity in animal models and
describe the potential usefulness of this technology as a strategy for HCV vaccine
design and immunotherapy.

INTRODUCTION
The inhibition of virus replication by the immune system is of paramount
importance to limit the spread of infection and moderate the course of the disease.
Essentially, control of viral infections consists of non-specific innate immune
responses and adaptive responses to viral specific proteins (Parkin and Cohen,
2001). Vaccine intervention aims to stimulate B cell antibody production (humoral)
and cell mediated (CD4+ and CD8+) immunity (Begue, 2001b). However, there
have been several major obstacles that have hampered the development of an
effective HCV vaccine. Firstly, apart from humans, the only infectious HCV
animal model is the chimpanzee, a protected species that is costly and hence
limited in its availability (Bukh, 2004). Secondly, although extensive studies from
chimpanzees and patients have provided insights into immune responses, it remains

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unclear as to why the immune system, in many cases, is inefficient in eliminating

HCV infection (Gremion and Cerny, 2005). Despite detectable humoral and cell
mediated immunity, HCV induces 50-80% chronicity of infected individuals
leading potentially to cirrhosis and hepatocellular carcinoma (Harris et al., 2002).
HCV is also highly variable, divided into at least 6 major genotypes and more than
50 subtypes based on nucleotide diversity within core, E1 and NS5 genes (Zein,
2000). In addition, as observed with other RNA viruses, HCV exists as groups
of related but distinct viral populations termed quasispecies variants that differ
in sequence diversity within distinct hypervariable regions along the genome.
The N-terminus of the E2 glycoprotein (a prime vaccine target) contains a major
hypervariable region 1 (HVR-1) and sequence variation in the HVR1 has been
associated with immunologically driven HVR1-antibody escape mutants (Farci et
al., 1997; Majid et al., 1999). The first neutralizing antibody epitopes were located
within the HVR-1 region, and hyperimmune serum obtained from immunization
of a rabbit with HVR-1 peptide has been found to protect against homologous
HCV in chimpanzees, but did not protect against 'escape' mutants that persisted
during chronic infection (Farci et al., 1996). Nevertheless, studies associated with
intravenous drug users who resolved previous HCV infection and who are less
likely to be re-infected suggests that immunity against HCV can be successfully
generated in some individuals (Mehta et al., 2002). Furthermore, recent work
illustrates that targeting multiple epitopes in the HCV envelope proteins (E1 and
E2) may facilitate a more broad neutralization capacity (Bartosch et al., 2003a).
Immunological studies of acute phase HCV infection suggest that this period is
critical for determining the outcome of infection (Thimme et al., 2001). In humans
and chimpanzees, the clearance of acute infection is accompanied by a strong and
multi-specific CD4+ and CD8+ T cell response (Haefelin-Neumann et al., 2005;
Rehermann and Nascimbeni, 2005). Immune mediated control of HCV infection
is also evident from intrahepatic compartmentalization of HCV specific T cells,
aggressive disease progression in HCV/HIV co-infected individuals and increased
viral loads with immunosuppression of patients (Gremion and Cerny, 2005).

Here, we examine the problems encountered in the development of HCV vaccines

and evaluate current as well as future vaccine strategies to generate effective immune
responses to HCV. Several approaches designed to elicit immune responses to HCV
structural proteins (Core, E1 and E2 glycoproteins) and the bi-functional viral
serine protease/helicase (NS3) that contains conserved 'immunodominant' regions,
have been tested in non-human primates, monkeys and murine models. Although
a comprehensive review of these technologies is beyond the scope of this chapter,
we briefly describe the need for more optimal approaches to HCV vaccine design.
In this regard, we discuss the recombinant Vesicular Stomatitis Virus (VSV) as a
vector that could be useful in the fight against HCV infection.

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CLASSICAL STRATEGIES IN VIRAL VACCINES

The principle of vaccination is to induce a 'primed' state in the host so that infection
with a pathogen will result in a rapid secondary immune response. The goal to
eliminate or inhibit replication of the organism and protect from clinical disease is
dependent upon memory T and B cells as well as neutralizing antibody in the serum.
Virus infection and replication in host cells is known to elicit long-lived antibody
and cell-mediated immunity. These features make viral vectors attractive as vaccine
candidates after safety issues are addressed by attenuation or inactivation, since
they can often induce long-term immunity following a single dose.

Non-virulent viral vaccine strategies have been successfully developed for a number
of viral pathogens that can be grown in cell culture to facilitate their attenuation or
inactivation (Begue, 2001b, Gershon, 1990; Hinman and Orenstein, 1990; Matter,
1997). Live attenuated viruses typically have reduced virulence caused by repetitive
passage during in vitro cell culture growth conditions. Selected mutants replicate
poorly in the host and do not cause disease but efficiently induce long-lived antibody
and cell-mediated immunity. Indeed, Measles, Mumps and Rubella (MMR vaccine)
are controlled in many developed countries through this live attenuated vaccine
approach (Burgess, 1994; Wharton et al., 1990; Zimmerman and Burns, 1994).
The worldwide eradication of smallpox is another example of a live attenuated
heterologous vaccine. In this case, the cross reacting immunity of vaccinia (less
virulent) is protective against variola virus, the causative agent of small pox (Begue,
2001a, Enders et al., 2002). Selectively targeted live attenuated vaccines also
include single doses of yellow fever for travellers and varicella-zoster virus for
the elderly (Hill, 1992; Marfin et al., 2005; Senterre, 2004; Takahashi, 2004). The
main drawback of live attenuated vaccines however, is the danger of reversion to
virulence and the possibility of causing extensive disease in immunocompromised
individuals.

When live attenuated vaccines are unavailable, inactivated preparations of the

virulent organism using beta-propiolactone or formaldeheyde are an option
(Bachmann et al., 1993; Jiang et al., 1986; King, 1991). However, these inactivated
vaccines generally only stimulate humoral responses, are expensive to prepare and
in addition, the chemical inactivation can directly impair certain immune responses
such as T cell activation (Bachmann et al., 1993). However, the Salk poliovirus
vaccine containing all 3 polio-virus strains is a successful example of this approach
and is particularly useful in protecting immuno-suppressed children (Pearce, 2004).
The live, less expensive Sabin polio-vaccine has been adopted in many parts of the
world due to lower costs and elicits effective induction of mucosal immunity (Pearce,
2004). Seasonal Influenza vaccines similarly comprise inactivated Influenza A and
B strains (Hill, 1992; Marfin et al., 2005; Schwartz and Gellin, 2005; Takahashi,
2004). For Rabies virus, a human diploid cell culture-derived inactivated vaccine

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is administered either for post exposure prophylaxis following a rabid animal bite
or pre-exposure prophylaxis to protect animal workers at risk of infection from
occupatioal exposure (Lodmell and Ewalt, 2004).

In regard to other major etiological agents of liver disease, effective hepatitis A

virus formalin inactivated cell culture vaccines are available (Provost et al., 1986;
Strader and Seeff, 1996). Two doses administered one month apart appear to induce
high levels of neutralizing antibodies. For hepatitis B virus (HBV), HBV surface
protein purified from viral carriers, or a recombinant viral approach have been
successfully utilized to protect against HBV infection (Coursaget et al., 1990; Goilav
and Piot, 1989; Magnani et al., 1989; Prince et al., 1984; Szmuness, 1979). In the
first strategy, a trial in homo-sexual men in the USA showed that 3 intramuscular
injections at 0, 1 and 6 months appeared to protect 95% of vaccinees (Goilav and
Piot, 1989). However, the latter HBV vaccine is now more widely used in the
universal childhood immunization scheme and is given at 6, 10, and 14 weeks of
age, and likely requires a booster later in life (Milne et al., 1992; Miskovsky et al.,
1991; Murata et al., 1989). Importantly, HAV and HBV vaccination demonstrates
the plausibility of protection against hepato-pathogens that replicate primarily in
immune-compromised environment of the liver (Willberg et al., 2003). However,
it has been more difficult to develop HCV vaccine along similar lines since HCV
does not replicate efficiently in cell culture. Recently, three independent reports
(see chapter 16) describe complete HCV replication of a chimeric genotype 2a
replicon in cell culture, which may facilitate vaccine strategies (Lindenbach et al.,
2005). Thus, large scale purification of chemically inactivated or attenuated HCV
strains using this technology remains an exciting prospect for HCV vaccine studies.
Meanwhile, alternative recombinant approaches to engineer immune responses to
HCV have currently been used in HCV vaccine studies.

STUDIES OF RECOMBINANT HCV PROTEIN SUB-UNITS IN

CHIMPANZEES
In the early 1990s, chimpanzees were immunized with purified recombinant HCV
E1 and E2 glycoproteins as these were presumed targets for virus neutralization.
The source vectors for these subunit vaccines were recombinant vaccinia (rVV)
since rVV expressed products were known to elicit potent antibody responses in
alternate vaccine regimens (Choo et al., 1994; Ralston et al., 1993). These rVV
vectors demonstrated high-level protein expression from prototype HCV-1 structural
(core-E1-E2 1-906) cDNA (Ralston et al., 1993). Purified E1 and E2 products mixed
with the MF59 adjuvant also produced neutralizing antibodies since protection
was observed following challenge with low doses of homologous HCV but not
against heterologous HCV infection (Choo et al., 1994; Houghton et al., 1997; Ott
et al., 1995). Subsequently, since E1/E2 antigens appeared susceptible to genetic
variation amongst HCV types, the HCV core protein that is highly conserved

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amongst HCV genotypes was also evaluated. However, since subunit vaccines
alone appear inefficient at inducing cytotoxic T lymphocyte (CTL) responses, the
core protein was combined with a 40nm matrix composed of saponins, cholesterol
and phospholipids (ISCOM) (Polakos et al., 2001). The classical ISCOM method
entraps the antigen inside the adjuvant platform to facilitate priming of CD4+
and CD8+ mediated responses (Takahashi et al., 1990). For HCV vaccine studies,
a non-classical ISCOM approach was used where E. coli purified core protein
was adsorbed onto ISCOMATRIX. The resulting particulates stimulated strong
long-lived, CD4+ and CD8+ responses and induced Th0-type (Th1 and Th2-type
cytokines) as well as anti-core antibodies in Rhesus Macaques (Polakos et al., 2001).
The prospects of HCV ISCOM-vaccines to produce sterilizing immunity awaits to
be reported but a core vaccine itself may have therapeutic value since core-specific
CTLs in HLA-B44+ patients co-incided with lower viral titers, and core-CD4+
T cell responses correlated with milder courses of liver disease (Bottarelli et al.,
1993; Hiroishi et al., 2004)

To faciliate broader responses to HCV types, other studies have utilized truncated
forms of E1aa192-330 and E2aa 390-683 (HCV-N2) purified from baculovirus-infected
cells combined with HVR-1 peptides from different isolates (HCV-6) (Esumi et al.,
1999). However, despite high antibody responses to E1/E2 in chimpanzees, the low
level immune responses to HVR-1 peptides resulted in lack of sterilizing immunity
to HCV-6 challenge that was achieved only by boosting HVR-1 (HCV-6) antibody
responses. It seemed that antibody responses alone were incapable of neutralizing
HCV infection and these observations pointed to the requirement for technologies
that may facilitate broader immune responses to control HCV infection. In this
regard, the introduction of DNA vaccination technologies offered alternative or
complementary approaches to E1/E2 subunit vaccines.

EFFICACY OF RECOMBINANT DNA APPROACHES IN

GENERATING HCV IMMUNITY
Naked DNA or plasmid vaccines encode viral genes that following inoculation of a
host are expressed to stimulate immune responses. DNA vaccination was reported to
prime good antibody responses and offer the advantage of increasing CTL responses
(Donnelly et al., 1997). In fact, DNA-derived immunogens encoding HCV core
and E2 sequences alone or fused with hepatitis B surface antigen induced antibody
and CTL responses to HCV or HBV in BALB/c mice (Inchauspe et al., 1997).
However, the recombinant DNA-vaccinated animals lacked neutralizing antibodies
that could block binding of purified HCV E2 to the putative cellular receptor, CD81
(Heile et al., 2000; Pileri et al., 1998). Furthermore, the same study highlighted
that endoplasmic retained recombinant E2 protein expressed in mammalian cells
was superior in eliciting neutralizing antibodies. The latter finding stresses the
importance of generating antigens that are correctly proteolytically processed and
therefore authentically folded (discussed below).

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Combining DNA vaccination with boosts of purified protein or recombinant viruses

expressing the same antigens appear to enhance immune responses (Pancholi et
al., 2000; Pancholi et al., 2003; Song et al., 2000). However, in order to assess the
effectiveness of immune responses generated using these and other HCV vaccine
approaches, surrogate HCV challenge technologies have been created to replace
the need for expensive chimpanzee models in preliminary studies. Two examples
include: vaccinia-expressing HCV structural proteins, or Listeria monocytogenes-
expressing HCV NS3, both of which were developed to monitor the efficacy of
elicited immune responses in murine and potentially higher animal models (Pancholi
et al., 2003; Simon et al., 2003). The HLA-A2.1 transgenic mouse model has been
especially useful for refining cell-mediated HCV immunity (Pascolo et al., 1997).
These mice are devoid of murine MHC-I molecules and are transgenic for human
HLA-A2.1 (A0201) monochain major histocompatability class I molecule. Studies
have shown that these mice recognized the same HCV-derived peptides found in
human HLA-A2.1-restricted CTLs (Arichi et al., 2000; Brinster et al., 2001). DNA
vaccines can be positively modulated as shown in murine studies with adjuvants
such as CpG motifs and Quil A that appear to induce Th1-(IL2 and IFNγ cytokine
profile) biased immune responses in addition to strong antibody responses as
demonstrated in DNA vaccination to HCV NS3 (Hong et al., 2004). Furthermore,
efficient presentation and priming of cell-mediated responses can be optimized
using cationic microparticles that carry DNA-based vaccines (Hagan et al., 2004).
Alternatively, recombinant semliki forest virus (rSFV) particles expressing NS3, in
combination with DNA vaccination or alone, have been found to stimulate strong
immune responses in mice (Brinster et al., 2002). The rSFV particles infect host
cells but do not replicate. However they express high levels of NS3, which appear
to induce (as with DNA vaccine) NS3-specific CTLs targeted to dominant HLA-A2
epitopes described in patients (Urbani et al., 2001). However, only rSFV-NS3/DNA
combination experiments elicited anti-NS3 antibodies in non-transgenic BALB/c
mice (Brinster et al., 2002). Unfortunately, in chimpanzees, DNA immunization-
encoding HCV E2 appeared incapable of generating sufficient antibody and cellular
immune responses to clear HCV infection (Xavier et al., 2000). A good HCV
vaccine should elicit strong antibody and cellular immune responses., and current
studies in HCV-DNA vaccine approaches suggest combinatory vaccine strategies
are likely required to enhance HCV antigen responses.

Advancements in recombinant viral technologies have led to additional strategies

that may generate safer approaches to viral based vaccine regimens. These include
production of virus-like particles (VLPs) that closely resemble properties of the
native virion. These VLPs have been shown to elicit potent humoral and importantly
cellular (CTL) activity as demonstrated with human papillomavirus-VLPs,
recombinant HIV and HBV VLPs in animal studies (Kahn et al., 2001; Roberts et
al., 1999; Roberts et al., 1998; Roberts et al., 2004; Rose et al., 2001). Recently,

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HCV-like particles based on C, E1 and E2 that are capable of stimulating CTL and
humoral activity have also been reported (see discussion below).

HEPATITIS C VIRUS-LIKE PARTICLES

Recombinant baculovirus expression of the HCV structural proteins (core/E1/
E2) has been reported to generate HCV-like particles (HCV-VLPs) that have
biophysical, ultrastructural, and antigenic properties similar to those of the putative
virions (Baumert et al., 1998; Baumert et al., 1999). Immunization of BALB/c
mouse strains or human HLA-A2 transgenic (AAD) mice with these 40-50nm
non-infectious HCV-VLPs suggests that they are efficient at generating broad and
vigorous humoral and cellular immune responses compared to immunization with
DNA (Murata et al., 2003). Mice vaccinated with HCV-VLPs developed antibodies
to HCV E1/E2. These anti-HCV E1/E2 antibody responses were enhanced by HCV-
VLP plus monophosphoryl lipid A [MPL] and QS21 adjuvant (AS01B) or CpG
10105 and especially with combination of AS01B plus CpG 10105 (Qiao et al.,
2003). Furthermore, isotype analysis of the induced anti-HCV envelope proteins
demonstrated that HCV-VLP alone induced immunoglobulin (Ig) G1 response while
the use of adjuvants ASO1B and CpG 10105 combined facilitated predominately
IgG2a response that is indicative of a Th1 response proposed to be important
for HCV clearance. The neutralization capacity of these anti-E1/E2 antibodies
induced by HCV-VLP cannot be tested easily due to the lack of a suitable small-
animal model system. However, by challenging mice with recombinant vaccinia
expressing HCV structural antigens (vv.HCV.S-genotype 1b), investigators were
able to examine the induction of immune protection in murine and higher animal
models (Jeong et al., 2004; Murata et al., 2003). Although vvHCV.S infection is not
representative of natural HCV infection, studies analyzing immunization strategies
with baculovirus generated HCV-VLPs showed that HCV-VLP vaccinated mice
were better protected against vvHCV.S than DNA immunization (Murata et al.,
2003). This protection is due in part to the fact that, as compared with animals
immunized with DNA methods, HCV-VLPs elicit strong CTL responses and
vigorous CD8+ T cell responses against HCV core and E2 proteins. It appears
that unlike recombinant protein subunit vaccines, these virion like structures can
possibly be processed efficiently through the major histocompatibility complex 1
pathway and subsequently effectively prime CD8+ responses. HCV-VLPs therefore
offer promise for further study in chimpanzee or human trials.

Recently, infectious particle systems have also been described where pseudo-
particles are assembled that display unmodified and functional HCV glycoproteins
onto retroviral and lentiviral core particles (Bartosch et al., 2003b, Flint et al.,
2004). These particles were primarily designed to understand HCV cell entry but
could provide useful information for vaccine development especially with regard
to the potential neutralization of HCV glycoproteins to their target receptors using

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anti-sera of animals treated with different vaccine regimens. In fact, the presence
of a green fluoresent protein marker packaged within these HCV-pseudotype
allowed determination of infectivity mediated by the HCV glycoproteins in primary
hepatocytes and hepato-carcinoma cells (Bartosch et al., 2003b). This infectivity
was neutralized using patient sera and by some anti-E2 monoclonal antibodies,
indicating a role for neutralizing antibodies against HCV glycoproteins. The
potential modification of these particles to render them non-infectious may allow
for in vivo vaccine studies in animal models.

In addition to the above studies, our laboratory and others have focused on using
vesicular stomatitis virus (VSV) as a candidate for evaluation as a virus-based
strategy for HCV vaccination and/or immuntherapeutic studies (Ezelle et al., 2002;
Majid et al., 2005). The advantages of using VSV-based approaches to generate
immune responses against HCV are discussed below.

VSV AS A SAFE AND POTENT VECTOR FOR GENERATING

IMMUNITY AGAINST VIRAL PATHOGENS
VSV is a member of the Rhabdoviridae family and is a negative-stranded cytopathic
virus. Rhabdoviruses are classed into at least 5 genera. Vesiculoviruses, Lyssaviruses
and Ephemeroviruses infect animals (both vertebrates and invertebrates), whereas
Cytorhabdoviruses and Nucleorhabdoviruses infect plants. VSV is composed of an
11-kilobase negative-sense RNA genome and is relatively simple since it encodes
for only five viral proteins: the nucleocapsid (N) that encases the virus genome, 2
polymerase proteins (L and P), a single surface glycoprotein (G) and a peripheral
matrix protein (M). Rhabdoviruses are enveloped viruses and their glycoproteins
are classed as typical type 1 membrane proteins consisting of polypeptide trimers
of the viral G protein (Coll, 1995). This G protein is a dominant viral antigen and is
responsible for viral infection through unidentified cellular receptor(s) on a variety
of mammalian and insect cells, and has been shown to be a target for neutralizing
antibody (Beebe and Cooper, 1981; Chan et al., 1982; Hardgrave et al., 1993;
Lefrancois and Lyles, 1983).

There are several features that make VSV an excellent candidate as a vaccine
vector. This weak human pathogen does not undergo genetic recombination or
genomic reassortment and has no known transforming properties. Furthermore,
VSV does not integrate any of its genomic material into host cell DNA (Barber,
2004; Lawson et al., 1995; McKenna et al., 2003). As a vaccine strategy, VSV
is known to elicit strong humoral and cellular immune responses in vivo and
naturally infects at mucosal surfaces (Haglund et al., 2002; Martinez et al., 2004;
McKenna et al., 2003). This feature offers an alternative less invasive intranasal
route of immunization that has been shown to induce both mucosal and systemic
immunity (Kahn et al., 2001; Reuter et al., 2002; Roberts et al., 1999). In addition,

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recombinant VSVs can be generated to accommodate large foreign gene inserts

or multiple genes into their genomes (Ezelle et al., 2002; Fernandez et al., 2002;
Obuchi et al., 2003). Other RNA viral vectors, such as those based on alphaviruses
and poliovirus, do not typically tolerate incorporation of large foreign genes to form
replication-competent RNA viruses (Falkner and Holzer, 2004; Schlesinger, 2001).
Furthermore, VSV grows to high titers in vitro, thus facilitating rapid purification
of large amounts of virus and viral proteins.

However, natural VSV infection has been reported in cattle, horses and swine
causing significant disease, including vesicular lesions around the mouth, hoofs, and
teats with loss of beef and milk production (Martinez and Wertz, 2005). In the US,
livestock is periodically infected with one of two serotypes of VSV (Indiana, VSVI
or New Jersey, VSVNJ). VSV infection is asymptomatic in humans but in rare cases,
chills, myalgia and nausea have been reported (Coll, 1995; Fields and Hawkins,
1967; Johnson et al., 1966; Wagner, 1996). As a consequence of rare infectivity in
humans, seroprevalence of VSV antibodies within the general population is low
except in limited regions in Georgia (VSV NJ ) or Central America (VSV I and VSV
NJ ). Antibodies are also detected in individuals who have high risk of exposure such
as laboratory workers, veterinarians and ranchers (Johnson et al., 1966). This low
VSV seropositivity in the general population and the lack of serious pathogenicity
in humans are advantages in the potential use of live recombinant VSV-vectored
vaccines in humans.

VSV-HCV PSEUDOTYPES AS A PRELUDE TO RECOMBINANT

VSV IN HCV IMMUNO-INTERVENTION
Since VSV was known to infect many cell types and can incorporate foreign
viral glycoproteins on its surface, it was used first in HCV research as a tool to
generate reagents to understand HCV infection in cell culture. The use of VSV in
HCV research initially focused in generating pseudotype viruses expressing HCV
envelope proteins on their surface (Matsuura et al., 2001; Meyer et al., 2000). These
recombinant VSV pseudotypes were used to understand HCV cell entry in the
absence of conventional cell culture systems. Several lines of evidence using VSV-
HCV pseudotypes pointed to functional role for both E1 and E2 glycoproteins in
pseudotype virus infectivity. First, sera derived from chimpanzees immunized with
homologous HCV envelope glycoproteins were found to neutralize virus infectivity.
Secondly, as with many pH-dependent enveloped viruses, VSV-HCV pseudotype
entry was negatively influenced by low pH pre-treatment in a number of susceptible
cell lines (Meyer et al., 2000). The same study also demonstrated that Concavalin
A (plant lectin) neutralized both E1 and E2 pseudotype virus infectivity. These
findings illustrated that carbohydrate structures on HCV envelope glycoproteins may
influence binding of HCV to cells directly or by attachment to carbohydrate binding
proteins for attachment to or infection of cells. Furthermore, the use of monoclonal

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antibodies to block putative HCV cell entry receptors, CD81 or LDL, appeared to
reduce pseudotype plaque titers demonstrating specificity of VSV-HCV pseudotype
binding to cells (Agnello et al., 1999; Cormier et al., 2004; Matsuura et al., 2001;
Meyer et al., 2000; Pileri et al., 1998). However, technical limitations in generating
VSV-HCV pseudotypes resulted in expression of either HCV E1 or E2 alone, and
not as a functional E1/E2 non-covalently linked glycoprotein complex as found in
natural HCV infection (Beek et al., 2004). Furthermore, the chimeric nature of the
recombinant HCV E1 or E2 fused to the cytoplasmic tail of VSV glycoprotein may
influence interpretation of the results (Meyer et al., 2000). However, others also
showed that VSV-HCV pseudotypes could be generated that possesed chimeric E1
or E2 glycoproteins either individually or together (Matsuura et al., 2001). In their
report, VSV glycoprotein was replaced with the green fluorescent protein (GFP) and
infectivity of pseudotypes was determined by GFP-expressing cells. Importantly,
their study illustrated that co-expression of both HCV glycoproteins in the VSV-
pseudotypes was required for maximal infectivity. Subsequently, we and others
adapted an alternative VSV approach that utilized the genetic manipulation of the
full length VSV genome to generate novel reagents for vaccine strategies.

REPLICATION COMPETENT AND DEFECTIVE RECOMBINANT

VSV AS A VACCINE STRATEGY TO GENERATE IMMUNE
RESPONSES
The availability of recombinant DNA technology has allowed for genetic
manipulation of the VSV genome and recovery of infectious VSV entirely from
cDNA clones (Lawson et al., 1995). Rabies virus was the first Rhabdovirus to be
recovered from a complete cDNA clone (Schnell et al., 1994). An important quality
of the approach utilized the initiation of the infectious cycle by expressing the
antigenomic RNA rather than the genomic RNA in cells expressing the viral N, P,
and L proteins. This strategy avoids potential anti-sense problems effecting viral
replication in which mRNAs encoding the N, P, and L proteins would hybridize
to the negative-strand genomic RNA. This same approach has been successfully
applied to full-length positive-strand cDNA for VSV. The high recovery of VSV
using this approach combined with the genetic malleability of the VSV cDNA has
useful applications to vaccine development. Gene expression of the non-segmented
negative-strand RNA viruses is controlled by the highly conserved order of genes
relative to the single transcriptional promoter (Wagner, 1996). The rearrangement
of these genes appears to affect virus phenotype and although live attenuated
viruses can be generated, they appear to have reduced capacity to generate clinical
disease in the natural hosts such as the domestic swine (Flanagan et al., 2001).
Nevertheless, these attenuated viruses were still effective delivery mechanisms
for generating VSV-G immune responses and protection against wild type VSV
following challenge of vaccinated animals (Flanagan et al., 2000; Flanagan et al.,
2001).

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In subsequent studies, it was demonstrated that by introducing an extra foreign

transcription unit between G and L proteins, rVSVs could be recovered that
highly expressed and efficiently incorporated foreign proteins into their virions
(McKenna et al., 2003; Schnell et al., 1996). These include CD4 receptor, measles
virus glycoprotein (Schnell et al., 1996), and influenza virus hemagglutinin (HA)
and neuraminidase (NA) (Kretzschmar et al., 1997). Furthermore, EM analysis
of rVSV-HA/NA influenza particles demonstrated that the recombinants were
mosaics carrying both VSV-G and influenza glycoproteins. There appears therefore
significant space in the VSV membrane that can accommodate foreign membrane
proteins.

From the features described above it is clear that vaccines based on live VSV
recombinants have advantages over other live recombinant vaccine vectors. First,
compared to large complex genomes of the Poxviridae family that encode numerous
proteins that include immunoevasive and immunosuppressive proteins, the VSV
genome is relatively simple, well understood and easier to manipulate (Lawson
et al., 1995). Second, there is a potential for generating live attenuated viruses
with reduced pathogenic phenotypes (Flanagan et al., 2000). Third, compared to
segmented genomes of viruses in the Orthomyxoviridae family, the single-stranded
genome of VSV does not undergo re-assortment and therefore these attenuated
viruses cannot genetically recombine with wild-type viruses in vivo.

RECOMBINANT VSV VACCINE STUDIES

VSV infection can be neuropathic in mice, following high dose intranasal
infection, since olfactory receptors are highly tropic for VSV (Bi et al., 1995).
Lethal encephalitis can occur especially in young mice but the virus can also be
cleared by innate and adaptive immune responses (Balachandran et al., 2000a,
Durbin et al., 1996; Meraz et al., 1996; Muller et al., 1994). Several recombinant
VSV studies against human pathogens have been tested in murine models. For
example, antibodies to influenza glycoprotein HA appear to be neutralizing in
natural infection and a recombinant VSV-HA (rVSV-HA) has been shown to
successfully generate protective immunity to lethal doses of influenza A/WSN/33
(H1N1) virus in BALB/c strain of mice (Roberts et al., 1998). Furthermore, the
rVSV-HA was administered intranasally to elicit immune responses to the expressed
influenza HA protein. As described above, the rVSV cloned from cDNA appears to
have reduced pathogenicity compared to wild-type VSV Indiana strain. In related
studies, an attenuated live rVSV expressing influenza HA with a truncation of the
cytoplasmic domain of the VSV-G protein was intranasally administered to mice
and found to completely protect against influenza challenge (Roberts et al., 1999).
Similarly, in the same study a non-propagating rVSV devoid of VSV-G was also
capable of eliciting protective immunity to influenza HA. This vector was also
non-pathogenic and additional advantages over its replication counterpart include a

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lack of neutralizing antibody stimulation against the vector itself and the replication
defective strategy improves potential safety attributes as a viral vaccine vector.

In the case of respiratory syncitial virus (RSV), replication competent or attenuated

non-propagating VSV expressing RSV G (attachment) and RSV F (fusion)
glycoproteins has been reported (Kahn et al., 2001). The VSV-RSV-F virus appeared
to elicit RSV-specific antibodies in serum as well as neutralizing antibodies to RSV
that were found to be protective against RSV challenge in BALB/c mice.

Finally, the rVSV approach has also been successfully applied to higher animal
models. For example an AIDS vaccine based on attenuated VSV vectors expressing
SHIV env and gag genes was tested in rhesus monkeys (Rose et al., 2001). This
approach provided significant protection as demonstrated in vaccinated animals
challenged with pathogenic AIDS virus. Since VSV was shown to be a potent
inducer of cellular and humoral immunity in several viral models, we considered
VSV as a tool for delivering HCV immunity.

VSV EXPRESSING HCV CORE, E1 AND E2 STRUCTURAL

PROTEINS AS A POTENTIAL VACCINE AND
IMMUNOTHERAPEUTIC STRATEGY AGAINST HCV INFECTION
In our laboratory we have generated VSV-based HCV vaccine vectors that express
all the HCV structural proteins, to maximize an immune response to multiple HCV
epitopes (Ezelle et al., 2002; Majid et al., 2006). In the first strategy we developed
a recombinant live or replication competent VSV antigen delivery system (Ezelle
et al., 2002). To accomplish this, the contigous HCV core, E1 and E2aa 1-746 (HCV

Table 1. CTL activation by VSV-HCV-C/E1/E2 following intravenous injection. IFNγ

ELISPOT analysis was determined by ELISPOT. Splenocytes were harvested from
intravenously vaccinated BALB/c mice 4 weeks after initial injection and pulsed against
core, E1, or E2 peptides shown. IFNγ production is illustrated in VSV-HCV-C/E1/E2
(VSV-C/E1/E2) mice (Ezelle et al., 2002).

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Fig. 1. Construction of rVSV (VSV-HCV-C/E1/E2) expressing HCV Core, E1 and E2. HCV NIHJ1
(genotype 1b provided by T. Miyamura) Core, E1 and E2 regions (aa 1-746) were cloned into the
pVSV-XN2 vector (provided by J. Rose). Co-transfection of the recombinant pVSV-XN2-C/E1/E2
with vectors pBL-N, pBL-P, and pBL-L into BHK cells previously infected with vaccinia expressing
T7 polymerase (v-TF7-3), results in transcription, translation and replication of VSV-HCV-C/E1/E2.
The infectious rVSV virions are released from the cell (Ezelle et al., 2002, Lawson et al., 1995).

genotype 1b) open reading frame (ORF) was cloned into the Indiana (VSVI)
cDNA backbone encoded from the plasmid pVSV-XN2 (Fig. 1). In order to
create recombinant virus, BHK cells are infected with vaccinia encoding the T7
polymerase (vTF7-3) that drives expression of the N, P and L proteins of VSV
encoded in pBL-N, pBL-P, and PBL-L plasmids respectively. Co-transfection of
the attenuated VSV cDNA genome vector (pVSV-XN2-C/E1/E2) in this set up
allows for efficient recovery of recombinant VSV (rVSV) expressing foreign genes
(as described above).

The methodology above allows for large scale preparations of replication competent
rVSV for cell and animal studies. Importantly, we observed only slight attenuation
in the growth properties of replication competent VSV-HCV-C/E1/E2 as compared
to wild type virus counterparts as shown in Fig. 2 (Ezelle et al., 2002). Furthermore,

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Fig. 2. Growth curve analysis of rVSV. VSV-HCV-C/E1/E2 demonstrates a similar growth rate to
VSV-GFP. Infections were undertaken at an MOI of 1 for 30 min. Cell medium was analyzed for
viral titers at 6, 12, 18, and 24hr post-infection by standard plaque assay (Balachandran et al., 2000b,
Ezelle et al., 2002).

VSV-HCV-C/E1/E2 expressed high levels of HCV antigen illustrated in Fig. 3 (Ezelle

et al., 2002). In recent studies, analysis using a panel of conformational sensitive
mouse and human antibodies illustrated that rVSV expressed authentically folded
non-covalently linked E1/E2 heterodimers (Ezelle et al., 2002; Majid et al., 2006).

Fig. 3. Expression of HCV core, E1, and E2. BHK cells were infected with VSV-HCV-C/E1/E2 (VSV-
C/E1/E2) or control wild type VSV (VSV-XN2) at an MOI of 1. Cell lysates were analyzed for HCV
protein expression by immunoblot analysis 18 hr post-infection. The results demonstrate that VSV
can be used to express high levels of HCV structural antigens (Ezelle et al., 2002).

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 HCV Vaccines and Recombinant VSV

Fig. 4. Humoral immunity generated

to HCV structural antigens using
VSV. (A) Sera were collected
from BALB/c mice vaccinated
by intravenous injection (6 mice
per group) with VSV-HCV-C/E1/
E2 (VSV-C/E1/E2), VSV-GFP
or PBS only. Anti-HCV-E2 was
detected only in VSV-HCV-C/E1/E2
group when analyzed in an ELISA
format against recombinant E2
protein (Ezelle et al., 2002). (B)
Intraperitoneal administration of
VSV-C/E1/E2 can also generate
humoral immunity to HCV E2 in
BALB/c animals.

The binding of these VSV expressed HCV glycoproteins to patient antibodies from
native HCV infection is an important potential recognition of these immunogens in
HCV vaccine design. Furthermore, intravenous or intraperitoneal immunization of
BALB/c mice with VSV-HCV-C/E1/E2 elicited potent secondary serum antibody
responses to HCV E2 as detected by ELISA demonstrated in Fig. 4 (Ezelle et al.,
2002). Vaccine regimens involved intravenous injections with VSV-HCV-C/E1/E2
or control VSV-GFP, or PBS followed by a secondary inoculation 2 weeks later.
The sera were tested for antibody responses on day 21 post initial injection. Our
data indicated that VSV-HCV-C/E1/E2 is an efficient vehicle for generating HCV
E2 antibodies and warrant further study to assess their capacity in neutralizing
HCV infection. In addition, we have observed that VSV-HCV-C/E1/E2-injected
mice produced strong HCV core antibodies indicating that immune responses to
all the HCV structural proteins were being successfully generated.

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Table 2. CTL activation by VSV-HCV-C/E1/E2 following intraperitoneal route of inoculation.

Splenocytes were harvested and analyzed for IFNγ production by ELISPOT 7 days following
the injection. The results indicate again that only VSV-HCV-C/E1/E2 (VSV-C/E1/E2)
vaccinated mice activate T-cells when pulsed with HCV peptides (Ezelle et al., 2002).

In addition to strong humoral responses, the generation of multispecific CTL

responses are proposed to be essential for clearance of HCV during acute infections
in humans and chimpanzees (Cooper et al., 1999; Erickson et al., 2001; Neumann-
Haefelin et al., 2005; Thimme et al., 2001). Therefore, to determine if VSV-HCV-C/
E1/E2 was able to generate CD8+ T cell responses, we investigated CTL responses
in splenocytes from vaccinated mice using IFN-γ ELISPOT analysis against HCV
genotype 1b peptides known to stimulate CTL activity in BALB/c models (Gordon
et al., 2000; Nishimura et al., 1999). The release of IFN-γ cytokine from CD8+ T
cells is an indication of Th1 type immunity, and our studies illustrated that VSV-
HCV-C/E1/E2 could indeed elicit Th1 type CTL responses against HCV core, E1
and E2 peptides 4 weeks after the initial injection as illustrated in Table 1 (Ezelle
et al., 2002).

We have also evaluated whether routes of inoculation using rVSV could affect the
strength and type of immunity generated against HCV antigens. Intraperitoneal
injections were performed using VSV-HCV-C/E1/E2, VSV-GFP or PBS. Serum
collected on day 28 was tested again in an ELISA format and demonstrated
significant anti-E2 antibody levels in VSV-HCV-C/E1/E2 injected mice shown
in Fig. 4B (Ezelle et al., 2002). Interestingly, splenocytes harvested 7 days after
injection demonstrated CTL responses to HCV structural peptides as shown by
IFN-γ ELISPOT and presented in Table 2 (Ezelle et al., 2002). Collectively, our
data indicate that rVSV expressing HCV antigens can stimulate potent humoral
and cellular immunity in animal models. Studies ongoing in our laboratory also
suggest that a non-propagating VSV strategy may also be a feasible option for
generating immunotheraputic intervention to combat HCV infection. Given the
genetic malleability of VSV, the possibility of generating a number of vectors that

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are safe and yet very effective at generating immune responses to HCV remain an
exciting prospect.

CONCLUSIONS
The window against the quest for developing HCV vaccines and immunotherapy
has certainly shortened as result of a wealth of knowledge distributed with regard
to the immune responses that appear to be hallmarks of acute HCV clearance
in chimpanzees and humans. In addition, understanding key targets for virus
neutralization, particularly the HCV glycoprotein complex will aid the design
of immunogens and generate effective immune responses potentially to epitopes
broadly conserved amongst viral types. Furthermore, it appears increasingly likely
that viral neutralization by antibody alone may not be sufficient in generating
effective immune responses against a prophylactic HCV vaccine approach.
Nevertheless, antibody responses alone using subunit vaccines appears to reduce
chronicity of HCV infection, suggesting a therapeutic role in preventing HCV related
liver disease that may account to decreased morbidity and mortality in patients.
Indeed, an HCV glycoprotein-based subunit vaccine trial in humans demonstrated
a potential immuno-therapeutic role for vaccination (Nevens et al., 2003).

The innovation of new technologies that can elicit potent humoral and cellular
responses to multi-specific HCV antigens is likely key to a first line of defense
against HCV infection. Several promising approaches have been described in
this work. However, many studies are in preliminary phases using small animal
models and their findings require confirmation in HCV animal models, such as the
chimpanzees. The ability to effectively 'tune' individual immune responses against
key HCV antigens; core, E1, E2 (and NS3) may provide an opportunity for the
immune system to prevent HCV infection before this virus can outpace the immune
system as described in chronically infected individuals (Willberg et al., 2003). In
this regard, the VSV system described in this report offers significant promise since
this recombinant viral approach appears to be a potent stimulator of HCV immunity
in the murine model. Furthermore, several features of this system, for example, the
malleability of the VSV genome, ability to recovery high titer replication competent
or non-propagating recombinant viruses, high level expression of foreign genes, and
lack of pathogenicity in humans makes VSV an exciting tool for further endeavors
in development of a vaccine against HCV infection.

FUTURE TRENDS
Although there are still technical and immunological obstacles to overcome,
many exciting technologies being tested may improve vaccine studies and also
immunotherapy. Essential antigen presenting cells that initiate the immunological
cascade, such as dendritic cells (DCs) have been proposed to be impaired during
HCV infection although initial reports have been controversial and recent studies

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suggest normal functions of circulating plamacytoid or myeloid DCs (Bain et al.,

2001; Longman et al., 2005; Sarobe et al., 2003). In any case, it may be possible to
overcome this defect by autologous transfusion of HCV antigen-loaded mature DC
(Gowans et al., 2004). In addition, self replicating cytopathic and non-cytopathic
replicon transfection of DCs ex-vivo, illustrates efficient processing of HCV
antigens and stimulates efficient priming of T-cell responses following transfusion
into murine models (Racanelli et al., 2004). Furthermore, HCV-VLP uptake and
presentation has also been demonstrated by human DC (Barth et al., 2005). Key
goals therefore include better antigen delivery platforms and priming of efficient
immune responses.

The non-propagating VSV approach offers enhanced safety for a recombinant viral
vector. This virus can infect many cells, including DC, and can effectively activate
unknown innate responses that will inevitably facilitate efficient transduction
of adaptive immunity (Balachandran et al., 2000a, Balachandran et al., 2004).
Furthermore, modification of these viruses to express cell specific markers for
targeting or cytokines for adjuvant purposes is plausible. Finally, discovery of novel
innate intracellular molecules that are involved in anti-viral host defense may also
be inserted into VSV-based vectors to further facilitate potency of vaccine against
virus infection (Balachandran et al., 2004). These exciting advancements and better
understanding of HCV immunology suggest an adventurous future in the quest for
prophylactic and or immunotherapy against HCV infection.

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Chapter 16

Development of an Infectious HCV Cell

Culture System
Takaji Wakita and Takanobu Kato

ABSTRACT
Hepatitis C virus (HCV) infection causes chronic liver diseases and is a health
problem worldwide. Despite the increasing demand for knowledge on viral
replication and pathogenesis, detailed examinations of the viral life cycle have been
hampered by the lack of efficient viral culture systems, owing in part to its narrow
host range. We isolated full-length HCV clone, JFH-1strain, from a fulminant
hepatitis C patient. The JFH-1strain fit into the cluster of genotype 2a with notable
deviations in the 5'-untranslated region (5'UTR), core, NS3 and NS5A regions,
and monoclonality of the hyper-variable region sequence. The JFH-1 subgenomic
replicon replicated efficiently in a variety of cell lines without acquiring adaptive
mutations in its genome. Transfection of in vitro transcribed full-length RNA into
Huh7 cells, efficient replication of JFH-1 RNA and secretion of recombinant viral
particles into culture medium. Importantly, secreted viral particles were infectious
for both cultured cells and a chimpanzee. Furthermore, infectivity for cultured cells
was improved by using permissive cell lines. This infectious HCV system provides
for the first time a powerful tool to study the full viral life cycle, to construct anti-
viral strategies and to develop effective vaccines.

INTRODUCTION
Efforts to understand the viral life cycle of hepatitis C virus (HCV) and to identify
effective antiviral agents have been hampered by the lack of an efficient cell culture
system for this virus. Many attempts to develop a system for HCV infection and
replication in cell culture have already been undertaken; in fact, some advances
have been reported (Bertolini et al., 1993; Ito et al., 1996; Mizutani et al., 1996;
Iacovacci et al., 1997; Fournier et al., 1998; Rumin et al., 1999; Ito et al., 2001;
Zhao et al., 2002; Zhu et al., 2003). However, the viral replication efficiencies
reported in these studies were modest, requiring detection by a reverse transcription
polymerase chain reaction (RT-PCR). We hypothesized that the replication ability
of HCV may differ among HCV clones. We therefore isolated an HCV clone, JFH-
1, from a fulminant hepatitis patient with HCV (Kato et al., 2001). JFH-1-derived
subgenomic replicon proved capable of higher replicative capacity in a variety of
cell lines, and production of infectious HCV particles in Huh7 cells.

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A CASE OF FULMINANT HEPATITIS ASSOCIATED WITH HCV

In 1999, we obtained sera from a fulminant hepatitis patient (Kato et al., 2001).
The 32-year-old male patient was admitted with general fatigue, high-grade fever,
and liver dysfunction. No evidence of prior liver disease was found, and the patient
had no history of drug or alcohol consumption. In the previous 6 months, he had
not received any blood transfusions, taken any drugs intravenously, undergone
acupuncture, nor had sexual contact with a known hepatitis virus carrier. This
patient showed high levels of serum aspartate aminotransferase and alanine
aminotransferase, low levels of the minimum prothrombin time value, and displayed
stage II encephalopathy. HCV RNA was detected by RT-PCR, and anti-HCV
antibody was negative. All other hepatitis viral markers, anti-HAV antibodies (IgG
and IgM), hepatitis B virus (HBV) markers (HBsAg, anti-HBs, HBeAg, anti-HBe,
anti-HBc and HBV-DNA), and GB virus-C RNA, were negative. Therefore, he
was diagnosed as having HCV-associated fulminant hepatitis. The infection route
of HCV was obscure. The patient showed high levels of viremia, 105 copies/ml
at admission and 104 copies/ml 25 days later. However, HCV was undetectable
at 65 days after admission, at which point anti-HCV antibody was positive. At
75 days after admission, his condition improved and he was discharged from the
hospital.

To investigate the role of strain-specific viral characteristics of HCV in fulminant

hepatitis, we isolated HCV RNA from the acute phase serum of this patient,
amplified it by RT-PCR, and determined the sequence of its entire genome.

SEQUENCE ANALYSIS OF JFH-1

The HCV clone isolated from the fulminant hepatitis patient, designated JFH-1,
was determined to be of genotype 2a. To compare genomic characteristics, we also
determined the entire genomic sequences of 6 HCV genotype 2a clones isolated from
6 chronic hepatitis patients (JCH-1 to -6). JFH-1 is 9,678 nucleotides (nt) in length
with a long open reading frame spanning nt 341-9439 coding for 3033 amino acids
(aa). Clones isolated from 6 chronic hepatitis patients, JCH-1 to -6, comprised 9681,
9677, 9678, 9676, 9691, and 9686 nt, respectively, and encoded either 3032 or 3033
aa. In phylogenetic analysis, JFH-1 clustered with other genotype 2a clones, but
showed slight deviation from clones isolated from chronic hepatitis patients, JCH-1
to -6, and HC-J6 (prototype of HCV genotype 2a, accession number is D00944)
(Fig. 1). To determine the degree of deviation in each subgenomic region or entire
genome, the ratios of mean genetic distances [ratio = mean genetic distance between
JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) in each subgenomic region or
entire genome / mean genetic distance among all 2a strains in each subgenomic
region or entire genome] were calculated. For nucleotide analysis of the entire
genomes, the mean genetic distances between JFH-1 and other 2a strains (JCH-1
to -6 and HC-J6) and among all 2a strains were calculated to be 0.1136±0.0073 and

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Fig. 1. Phylogenetic tree based on the entire HCV genome of for JFH-1, JCH-1 to -6 and representative
strains for which the entire genome has been reported. The number of nucleotide substitutions per site
at each position was estimated by the six-parameter method, and a phylogenetic tree was drawn using
the neighbor-joining method. The length of the horizontal bars indicates the number of nucleotide
substitutions per site.

0.0969±0.0140, respectively, with the ratio of mean genetic distances representing

the deviation of clone JFH-1 among genotype 2a clones being 1.173 (Table 1).
Among analyses of each subgenomic region, the 5'UTR showed the greatest ratio
of mean genetic distances, 1.387, and was identified as the region with the greatest
deviation. For amino acids, mean genetic distances of the entire genome between
JFH-1 and other 2a strains (JCH-1 to -6 and HC-J6) and among all 2a strains were
0.0918±0.0052 and 0.0716±0.0139, respectively, giving a ratio of mean genetic
distances of 1.282. Analyses of each the subgenomic region revealed greater
diversity for core, nonstructural (NS) 3, and NS5A, with mean genetic distance
ratios of 1.560, 1.464, and 1.596, respectively. The complexity of HCV infection
in the fulminant hepatitis patient was also assessed by determining the distribution
of quasispecies in the hyper-variable region (HVR). Sequences of the 20 amplified
clones of the envelope (E) 2 region were determined and the frequencies of these
sequences were examined at the two time points at days 1 and 23 after admission.
In the early point of acute phase (day 1 after admission), 17/20 HVR sequences
were identical and those of the other 3 clones showed a difference of only one aa
substitution. At the later time point of the acute phase (day 23 after admission), the
HCV clones were identical (20/20,). These data suggest that the HCV in this patient
showed lower complexity than that of the general viral population. Monoclonality
of the viral population has also been reported for another case of HCV related

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Table 1. Ratios of mean genetic distance for each subgenomic

region.
Region Nucleotide Amino Acid
5'UTR 1.387 NA**
Core 1.251 1.560
E1 0.986 0.940
E2 1.107 1.066
NS2 1.243 1.298
NS3 1.168 1.464
NS4A 1.249 1.044
NS4B 1.178 1.223
NS5A 1.222 1.596
NS5B 1.213 1.208
3'UTR 0.989 NA
Entire Genome 1.173 1.282
*Ratios were calculated using the mean genetic distances [ratio =
mean genetic distance between JFH-1 and other 2a strains (JCH-1 to
-6 and HC-J6) in each subgenomic region or entire genome / mean
genetic distance among all 2a strains in each subgenomic region or
entire genome] (Kato et al., 2001).
**NA, not available

fulminant hepatitis (Farci et al., 1996). Thus, we speculated that monoclonality of

the viral population is related to the development of fulminant hepatitis and that
the JFH-1 clone, especially in the 5' UTR, core, NS3 and NS5A regions, has some
specific viral characteristics related to fulminant hepatitis.

PREFERENTIAL PROCESSING FOR CORE PROTEIN OF JFH-1

Among the subgenomic regions of JFH-1, 5'UTR, core, NS3 and NS5A were
identified as deviated regions. Among these regions, core protein is known to form
the viral particle and also to regulate multiple functions in host cells (Moriya et al.,
1998; Ray et al., 2001; Watashi et al., 2003; see Chapter 3). During virus assembly,
the core protein undergoes two consecutive membrane-dependent cleavages, and
it develops into two forms, p23 and p21 (Liu et al., 1997). The p21 core protein
is cleaved from the endoplasmic reticulum-bound p23 core protein or the longer
precursor polyprotein by host signal peptide peptidase (McLauchlan et al., 2002).
The p21 core protein was predominantly observed in patient serum containing native
viral particles (Yasui et al., 1998). Thus, the p21 core protein is the mature and
stable form that accumulates in the cell and eventually constitutes the viral capsid.
We investigated the differences in p21 core protein production between JFH-1 and
the other genotype 2a clones isolated from chronic hepatitis patients (JCH-1 to -5)
(Kato et al., 2003a). Using the core or core-E1 expression vector of JFH-1 and
JCH-1, we found that JFH-1 could preferentially produce the p21 core protein in

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both in vitro translation assay and cell transfection assay with Huh7, HepG2 and
HeLa cells. Similar results were also obtained when comparing JFH-1 with the
other clones (JCH-2 to -5) isolated from chronic hepatitis patients. Investigations
with chimeric constructs revealed that differences in core protein processing depend
on the c-terminal region of the core protein. We identified 4 aa substitutions in this
region of the core protein between JFH-1 and the other clones isolated from chronic
hepatitis patients. Through experiments with mutation-introduced constructs, all 4
of these aa of JFH-1 were found to be responsible for the preferential production
of p21 core protein. Based on these findings, we suspected that JFH-1 may be able
to preferentially produce viral particles over other HCV clones.

REPLICATION CAPACITY OF JFH-1 AS A SUBGENOMIC

REPLICON
To investigate the function of the NS region of JFH-1, we constructed a subgenomic
replicon system using this clone. The HCV subgenomic replicon system has enabled
us to mimic HCV replication in Huh7 cells, and has been used as a tool in the study
of the mechanism of HCV replication (Lohmann et al., 1999; see Chapter 11).
JFH-1 showed higher colony formation efficiency that was approximately 500-
fold more efficient than the prototype Con-1 replicon and 50-fold more efficient
than the Con-1/NK5.1 replicon, which contains highly adaptive mutations (Kato
et al., 2003b). Furthermore, the JFH-1 replicon could replicate efficiently not only
in Huh7 cells, but also in other hepatocyte-derived cell lines, HepG2 and IMY-N9
cells (Date et al., 2004), and non- hepatocyte derived cell lines, HeLa and 293 cells
(Kato et al., 2005). This result may be attributed to the replication proficiency of
JFH-1. Importantly, the JFH-1 replicon did not require an adaptive mutation in order
to replicate in these cell lines. Most clones isolated from each of these cell lines
showed no or a few aa mutations in the HCV-derived replicon regions. Previously,
Bukh et al. (2002) demonstrated that HCV infection could not be achieved with
full-length HCV RNA containing multiple cell-culture adaptive mutations. Thus,
the higher replication capacity and the absence of adaptive mutations of JFH-1 may
be important for developing an infectious HCV system.

CONSTRUCTION OF FULL-LENGTH JFH-1 cDNA

Based on results obtained using subgenomic replicons, we found that the JFH-
1 strain replicates very efficiently in Huh7 cells, as shown not only by colony
formation assay with G418 selection, but also by transient replication assay (Kato
et al., 2003a; 2003b; Date et al., 2004; Kato et al., 2005). This suggests that the
JFH-1 genome can replicate autonomously in Huh7 cells without the help of G418
selection pressure and the development of adaptive mutations. Taking advantage
of the efficiency of the JFH-1 strain replication capacity, we planned to test the
replication of a full-length JFH-1 clone in Huh7 cells.

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Monoclonality is one of the specific characteristics of HCV strains in fulminant

hepatitis, and the JFH-1 strain, as confirmed by isolations made from other patients
(Farci et al., 1996; Kato et al., 2001). This characteristic is also advantageous in the
construction of consensus clones in the production of full-length cDNA because,
usually, HCV possess a wide variety of mutations called quasispecies (Martell et al.,
1992). Thus, it was necessary to inject 10 different clonal mixtures into a chimpanzee
to establish the first infectious clone for chimpanzee (Kolykhalov et al., 1997). On
the other hand, JFH-1 cDNA was cloned from RT-PCR fragments and, although
some sequence diversity was present, the aa sequences were highly conserved and
full-length HCV cDNA encoding the JFH-1 strain consensus sequence was easily
assembled by connecting the cloned PCR fragments (Kato et al., 2001; Wakita et
al., 2005). The T7 promoter sequence was inserted just upstream of the full-length
JFH-1 cDNA sequence, and full-length synthetic JFH-1 RNA was transcribed from
pJFH-1 by T7 RNA polymerase.

REPLICATION OF FULL-LENGTH JFH-1 RNA IN Huh7 CELLS

We first transfected in vitro transcribed full-length JFH-1 RNA into naïve Huh7
cells, which is the original cell line used for subgenomic replicon studies. As we
expected, full-length JFH-1 RNA replicated efficiently in the transfected cells, as
determined by Northern blot analysis (Wakita et al., 2005). Viral proteins produced
from replicated RNA were demonstrated by immunofluorescence and Western blot
analyses. Transfection of replication incompetent mutant RNA transcribed from
pJFH1/GND, in which GDD catalytic motif of NS5B was mutated to GND, into
Huh7 cells, however, did not lead to viral replication or protein production.

We expected to achieve replication of full-length RNA in transfected Huh7 cells

because full-length genotype1b RNA with adaptive mutation had been reported
to replicate in Huh7 cells and subgenomic replicons of the JFH-1 strain had been
shown to produce more colonies in Huh7 cells than genotype1b replicons (Ikeda et
al., 2002; Pietschmann et al., 2002; Kato et al., 2003b). However, it was difficult to
predict viral particle formation and secretion because these had not been achieved
by full-length HCV RNA transfection, even though RNA replication was observed
in the transfected Huh7 cells (Ikeda et al., 2002; Pietschmann et al., 2002). To
determine whether the viral particles were formed and secreted into the culture
medium from the full-length JFH-1 RNA transfected cells, we performed several
biological assays. First, we analyzed the density of secreted viral proteins and
viral RNA by sucrose density gradient. It has been reported that the supernatant
of full-length replicon RNA replicating cells of the Con1 strain secrete viral RNA
into culture medium; however, the density of viral RNA was found to have a very
similar culture medium density as that from subgenomic RNA replicating cells
(Pietschmann et al., 2002). We thus first analyzed an aliquot of culture supernatant
from full-length JFH-1 RNA transfected cells by sucrose density gradient. Following

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ultracentrifugation, 16 fractions were obtained from the bottom of the tube. Both
viral core protein and RNA were quantified using sensitive core ELISA (Aoyagi
et al., 1999) and RT-PCR with real-time detection, respectively (Takeuchi et al.,
1999). Interestingly, both core protein and RNA peaks occurred in the same fraction
(around 1.17 g/ml), a density greater than the one where subgenomic replicon cells
usually segregate. Next, we determined RNase sensitivity of these peaks, as the viral
RNA genome packed in the particles should be protected from RNase digestion in
culture. Culture medium from the transfected cells were RNase digested, followed
by density centrifugation. The profile analysis of the density peaks revealed that
RNase digestion did not change the density gradient distribution of both RNA and
core protein, indicating the viral genome was protected from nuclease digestion
(Wakita et al., 2005).

Next, we confirmed whether the envelope proteins were incorporated into secreted
viral particles. If the viral particles are properly and completely formed and secreted,
viral genome and core protein form a nucleocapsid and are surrounded by envelope
proteins (E1 and E2 proteins). To assay envelope proteins, we treated culture
medium with detergent to strip the envelope components from the viral particles.
Viral envelope usually comprises cellular membrane components such as lipids,
making the density of envelope lighter than that of the inner nucleocapsids. We
found that both core protein and RNA peak fractions became heavier (around 1.25
g/ml), indicating the removal of the lighter envelope components by the detergent
treatment. Furthermore, we demonstrated the incorporation of both E1 and E2
proteins by Western blot analysis of peak fractions of viral RNA after density
gradient centrifugation. We collected approximately 2.5 litter of culture medium
from transfected cell cultures. Culture medium was concentrated by ultrafiltration
and then by ultracentrifugation, and was then fractionated by sucrose density
gradient. Each collected fraction (counted from the bottom of the centrifuge tube)
was further concentrated by ultrafiltration. Concentrated fractions were separated by
SDS-PAGE and then transferred onto PVDF membrane. Core, E1 and E2 proteins
were detected on each fraction using specific antibodies. Thus, all the components
of the viral particle were detected in the same density gradient fraction, suggesting
proper viral particle formation and secretion. Finally, viral particles secreted into
the culture medium were visualized by immuno-electron microscope analysis using
anti-E2 monoclonal antibody. Viral particles were shown to be spherical, with an
outer diameter of about 55 nm (Wakita et al., 2005).

INFECTIVITY OF SECRETED VIRAL PARTICLES FROM JFH-1

TRANSFECTED CELLS
After having confirmed the presence of secreted viral particles, we were interested
in the infectivity level of secreted viral particles. We used double-chambered culture
plates equipped with polyester membrane (0.45 μm pore size) separating the inner

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Fig. 2. HCV RNA replication (a) and core protein production (b) in infected Huh7 cells. Culture
medium was collected from full-length JFH1 RNA-transfected cells and concentrated by ultrafiltration.
Naïve Huh7 were seeded 24 h before infection. Filtered (0.45-µm pore size) culture media was
inoculated for 2 h with periodic rocking. After inoculation, cells were washed with PBS and were
cultured in complete culture medium for another 12, 24, 48, 72, and 96 h. Experiments were performed
in triplicate, and mean titers (closed circles) SD (bars) are shown.

and outer chambers. Thus, substances smaller than this pore size, such as virus
particles, can diffuse across the membrane and populate both chambers. Full-length
JFH-1 RNA transfected cells were transferred to the inner chamber and naïve Huh7
cells were seeded in the outer chamber. A few days after the start of the experiment,
naïve Huh7 cells in the outer chamber were stained with anti-HCV antibodies to
confirm infection by secreted virus particles. To our surprise, a few cells were
positively stained, although at very low frequency. To confirm that infection occurred
for naïve Huh7 cells, we collected culture medium of transfected Huh7 cells, which
was subsequently cleared by low speed centrifugation and filtered through a disk
filter (0.45 μm-pore size). Naïve Huh7 cells were inoculated with the cleared free
virus in a culture plate for 3 hours. Inoculated cells were then washed with PBS
and cultured for another 48 h in complete medium. To increase infection efficiency,
culture medium was concentrated by ultrafiltration. Inoculation of concentrated
culture medium increased the numbers of infected cells, however, the efficiency
was still low at around 0.5% with Huh7 cells. Inoculated cells were harvested after
infection, and HCV RNA titer was determined by PCR with real time detection
(Fig. 2a). Only 1% of inoculated HCV RNA was adsorbed by inoculated cells and
HCV RNA copies in the infected cells were further decreased within 12 hours
after inoculation. However, RNA titer in the infected cells increased at 24 hours
after inoculation. Core protein expression measured in infected cells by sensitive
ELISA showed a decrease within 12 hours after inoculation and an increase at 24
hours after infection (Fig. 2b). These data clearly showed that viral particles were
infectious for Huh7 cells, although at low efficiency (Wakita et al., 2005).

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NEUTRALIZATION OF JFH-1 INFECTIVITY BY CD81 ANTIBODY

AND PATIENT SERA
CD81 has been identified as an E2 protein binding protein (Pileri et al., 1998).
We thus tested the effectiveness of anti-CD81 antibody for inhibiting infectivity
of culture supernatant for naïve Huh7 cells. Naïve Huh7 cells were treated with
10 μg/ml of anti-CD81 antibody at room temperature and then washed with PBS
followed by inoculation with culture medium from transfected cells (Wakita et
al. 2005). HCV RNA titer in the inoculated cells was inhibited more than 1 log,
indicating that infection by secreted viral particles is at least partially dependent on
a CD81-specific pathway. Further studies will be necessary to determine whether
CD81 is a sole receptor molecule involved in adsorption and internalization steps
or whether other molecules are also involved.

The neutralizing activity in chronically infected patient sera has been shown by
experiments using pseudotype virus harboring HCV envelope proteins (Bartosch et
al., 2003; Yu et al., 2005; Logvinoff et al., 2004). We also tested some patient sera for
neutralizing activity against JFH-1. To increase sensitivity of the assay, a bicistronic
replicon construct containing luciferase reporter was used (Wakita et al. 2005).
Indeed, patient serum tested positive for neutralization of virus infection proved to
contain some neutralizing antibodies against JFH-1 (Wakita et al., 2005).

IN VIVO INFECTIVITY OF JFH-1 CULTURE MEDIUM

To further confirm authenticity of the viral particles produced in our study, in vivo
infectivity was tested in a chimpanzee (Wakita et al., 2005). Electroporated culture
supernatant was harvested from the full-length JFH-1 RNA-transfected cells and
cleared by low-speed centrifugation and then passed through a 0.45-μm disk filter.
Control culture medium was prepared from the cells mixed with JFH-1 RNA, but
with the omission of electroporation pulse. A chimpanzee was first inoculated with
undiluted control culture medium, and no infection was observed. Then, 104 diluted
culture medium harvested from the transfected cell was used for inoculation, but
again, no infection developed. Six weeks later, 103 diluted culture medium was
inoculated in the same subject, and viremia was induced. Viral titer was low, with
the highest HCV RNA titer being 2.04x103 copies/ml. Furthermore, HCV infection
was cleared without any evidence of abnormal liver histology or elevation of liver-
specific enzymes or HCV-specific antibody seroconversions (Wakita et al., 2005).
Further investigation is necessary to determine whether the nonvirulent phenotype
is a characteristic of the JFH-1 strain.

PERMISSIVE CELLS FOR JFH-1 INFECTION

The infection efficiency of JFH-1 was quite limited as only a small percentage
of the inoculated cells appeared positive for HCV by antigen staining (Wakita
et al., 2005). To increase the infection efficiency, specific cell lines derived from

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Huh7 cells were analyzed by several groups. Huh7.5, which is one of the cured
cell lines established from original HCV replicon cell lines, supported high levels
of subgenomic HCV replication with Con1 and H77 strains (Blight et al., 2002).
Huh7.5.1 is a cell line derived from Huh7.5 (Zhong et al., 2005). Indeed, infectivity
of Huh7.5 or Huh7.5.1 cell line with JFH-1 was markedly increased (almost 100%)
compared to standard Huh7 cells (Lindenbach et al., 2005; Zhong et al., 2005).
Furthermore, Zhong and colleagues (2005) also were able to prevent in vitro-
produced virus from infecting Huh7.5.1 using an anti-CD81 antibody, whereas
Lindenbach and his coworkers (2005) accomplished this with Huh7.5 by using a
soluble recombinant CD81 fragment.

SUMMARY AND CONCLUDING REMARKS

Recombinant HCV particles were produced and secreted from JFH-1 RNA-
replicating cells, and the secreted viruses were infectious to both Huh7 cells and
a chimpanzee (Zhong et al., 2005; Lindenbach et al., 2005; Wakita et al., 2005).
Biophysical property analysis showed that cell culture-grown virus particles have
a density of about 1.15 – 1.17 g/ml, are spherical, and have an outer diameter of
about 55 nm (Wakita et al., 2005). Both the density and the overall diameter of the
particle are in agreement with a recent report describing the production of virus
particles with a DNA-based expression system (Heller et al., 2005). Infectivity can
be significantly neutralized by CD81-specific antibodies, supporting observations
that CD81 plays an important role in HCV cell entry made in HCV pseudo particles
(Zhong et al., 2005; Lindenbach et al., 2005; Wakita et al., 2005; Bartosh et al., 2003;
Hsu et al., 2003). Some level of neutralization was achieved with immunoglobulins
in patient serum, showing that potentially protective antibodies are generated during
chronic infection but that their capacity to prevent chronicity may be limited. We
also observed cross-neutralization in sera from patients infected with a genotype
1 virus (Wakita et al., 2005).

Thus, JFH-1 is the first HCV strain with the capability to produce infection in
tissue culture, and serves as a platform for a new generation of HCV investigations.
Furthermore, the use of permissive cell lines such as Huh7.5 and Huh7.5.1 cell lines
will further expedite full virus culture experiments in the laboratory. This infectious
HCV system should provide opportunities to study the full HCV life cycle, including
virus entry, replication, virus particle formation, and virus secretion, as well as to
develop effective antivirals and vaccines.

ACKNOWLEDGEMENTS
Analysis of immuno-electron microscope and neutralization of infectivity of JFH-
1 virus were done by Dr. Ralf Bartenschlager's group (University of Heidelberg,
Heidelberg, Germany). In vivo experiment using a chimpanzee was done by Dr. T.
Jake Liang's group (National Institute of Health, Bethesda, Maryland). Infection

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experiment using Huh7.5.1 cells was done by Dr. Frank Chisari's group (Scripps
Research Institute, La Jolla, California). Supported by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science and the Ministry of
Health, Labor, and Welfare of Japan, by the Program for Promotion of Fundamental
Studies in Health Sciences of the National Institute of Biomedical Innovation
(NIBIO), and by the Research on Health Sciences focusing on Drug Innovation
from the Japan Health Sciences Foundation.

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