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MEDICAL
INTELLIGENCE
UNIT 31
EUREKAH.COM
GEORGETOWN, TEXAS
U.S.A.
in memoria di
mia nonna
Elisabetta Michelotti Mattaini
n. 11 aprile 1902
Fiano, Italia
m. 7 agosto 2000
Bessemer, Michigan
e
mio nonno
Pietro Luigi Mattaini
n. 23 luglio 1897
Sesona di Vergiate, Italia
m. 17 giugno 1976
Bessemer, Michigan
GB
CONTENTS
Preface ............................................................................................................. 7
1. Introduction to Retroviruses and Retroviral Vectors .................................... 1
Gary L. Buchschacher, Jr.
Abstract ....................................................................................................... 1
Introduction ................................................................................................ 1
General Background and Classification of Retroviruses ................................ 1
The Retrovirus Genome and Virion Structure ............................................. 2
The Retrovirus Replication Cycle ................................................................ 3
Elements of Retroviral Vector Systems ......................................................... 6
Summary ..................................................................................................... 9
Acknowledgements .................................................................................... 10
EDITOR
Gary L. Buchschacher, Jr., M.D., Ph.D.
Division of Hematology/Oncology
Department of Medicine
University of California-San Diego
La Jolla, California, U.S.A.
Chapter 1
CONTRIBUTORS
Eric O. Freed
Laboratory of Molecular Microbiology
NIAID, NIH
Bethesda, Maryland, U.S.A
Chapter 2
Mehdi Gasmi
University of California-San Diego
La Jolla, California, U.S.A.
Chapter 6
James R. Gilbert
University of California-San Diego
La Jolla, California, U.S.A.
Chapter 5
John C. Kappes
Department of Medicine
University of Alabama at Birmingham
Birmingham, Alabama, U.S.A.
Chapter 8
John C. Olsen
Cystic Fibrosis/Pulmonary Research
and Treatment Center
University of North Carolina
Chapel Hill, North Carolina, U.S.A.
Chapter 7
Ina Roy
Center for Bioethics
University of Pennsylvania
Philadelphia, Pennsylvania, U.S.A.
Chapter 10
Sybille L. Sauter
GenStar Therapeutics
San Diego, California, U.S.A.
Chapter 6
Narasimhachar Srinivasakumar
Division of Hematology/Oncology
Department of Medicine
Vanderbilt University
Nashville, Tennessee, U.S.A.
Chapter 4
Marie A. Vodicka
Human Biology
Fred Hutchinson Cancer
Research Center
Seattle, Washington, U.S.A.
Chapter 3
Flossie Wong-Staal
University of California-San Diego
La Jolla, California, U.S.A.
Chapter 5
Xiaoyun Wu
Department of Medicine
University of Alabama at Birmingham
Birmingham, Alabama, U.S.A.
Chapter 8
Jiing-Kuan Yee
Department of Virology
Beckman Research Institute City
of Hope
Duarte, California, U.S.A.
Chapter 9
John A. Zaia
Department of Virology
Beckman Research Institute City
of Hope
Duarte, California, U.S.A.
Chapter 9
PREFACE
CHAPTER 1
Introduction to Retroviruses
and Retroviral Vectors
Gary L. Buchschacher, Jr.
Abstract
s various viral vector systems for gene transfer are developed, interest in using such
systems in applied settings continues to grow. This Chapter is designed to provide
background information for readers interested in learning about lentiviral vector systems for gene transfer applications but who lack a background in retrovirology. To assist those
readers who are unfamiliar with retroviral vector systems, basic outlines of the retroviral replication cycle and of characteristics of retroviral vector systems are introduced here in order to
present and define concepts and terms that are discussed in subsequent Chapters.
Introduction
The development of vector systems derived from the lentivirus genus of retroviruses has
potential for possible clinical gene transfer applications.1 Retroviral vector systems used previously in gene transfer applications have been derived from the oncogenic retrovirus genus. The
oncoretroviruses, sometimes (though not necessarily correctly) referred to as simpler retroviruses
in comparison to lentiviruses, had been studied for years and knowledge gained from those
studies enabled development of vector systems based on the parent viruses. The concepts governing development of oncoretrovirus-derived vector systems also guide the development of
lentiviral vector systems. Therefore, an understanding of the retroviral replication cycle and of
the basic features of retroviral vector systems based on oncogenic retroviruses is necessary in
order to fully understand the complexities and issues associated with the development of lentiviral
vector systems discussed later in this book.
In this first Chapter, the retroviral replication cycle and concepts related to development
of retroviral vector systems are outlined. Since a comprehensive review of the various retroviruses
and vectors derived from them is obviously beyond the scope of this section, the information
presented will describe generic features shared by retroviruses and vector systems in general,
rather than focus on one or more particular viruses. Readers who are familiar with these concepts are advised to skip this introductory Chapter and to move on to Chapter 2.
mouse mammary tumor virus (MMTV), and the avian pathogens Rous sarcoma virus (RSV),
spleen necrosis virus (SNV), and avian leukosis virus (ALV). These viruses were discovered and
were of interest because of their association with the development of tumors in their host
organisms. Study of these viruses eventually led to the discovery and development of the oncogene
theory of tumorigenesis: some of the viruses actually contained oncogenes within their genomes, while others interacted with oncogenes in either a direct or indirect way to contribute
to tumor formation.3,4
Over time, other retroviruses that had in common with the previously isolated viruses
many features of their genome organization and overall replication strategy were discovered.
Historically, because of their pattern of pathogenicity, these viruses were grouped into three
subfamilies: 1) the acutely oncogenic retroviruses, or oncoretroviruses (such as those described
above); 2) the lentiviruses (associated with slow diseases or those with long latent periods); 3)
the spumaviruses (foamy viruses, named because of the pathogenic changes observed in infected cells). Viruses were also grouped (Types A-D) according to the electron microscopic
appearance of their nucleocapsid structures.5 Further study of these viruses enabled detailed
comparison of their genome structures and nucleic acid sequences, which resulted in a further
refinement of the retrovirus classification system.6 This led to a revised classification of the
Retroviridae family into seven genera: mammalian type B retroviruses, mammalian type C
retroviruses, avian type C retroviruses, mammalian type D retroviruses, HTLV/BLV type
retroviruses, lentiviruses, and spumaviruses. The remainder of this Chapter will focus on describing general features of the retrovirus replication cycle and retroviral vector systems, focusing on simple oncoretroviruses. Although vector systems derived from MLV are the most commonly used retroviral-based systems in gene transfer applications, this discussion will not focus on
MLV per se, but will describe prototypical features characteristic of the retroviruses in general.
Fig. 1. Genome structure of a prototypical retrovirus. The genomic viral RNA, represented by a single black
line, is shown at the top of the figure, with the structure of the resulting provirus after reverse transcription
below. The locations of the open reading frames gag, pol, and env are shown. Reverse transcription of the
RNA results in rearrangement of the termini of the genome, resulting in the structures of the LTRs (long
terminal repeats) as indicated. Cis-acting sequences of the viral genome are shown in more detail in Figure 3.
polyadenylation signal located in the 3 LTR. The gag gene encodes viral core structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC). Some retroviruses also encode various other small proteins or peptides within the gag open reading frame. The pol gene encodes
the viral replication enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN).
The env gene encodes the envelope glycoprotein (Env) which is processed into transmembrane
(TM) and surface (SU) subunits.
Virion Structure
Retrovirus particles consist of the viral protein core and the surrounding viral envelope
that is made up of a cellular membrane-derived lipid bilayer and viral-encoded envelope glycoproteins. The structural core of the virion contains the two RNA molecules (associated as a
dimer) in association with the nucleocapsid protein that, in turn, is surrounded by a shell of
capsid protein. The matrix protein is located outside of the viral capsid and appears to interface
with the inner part of the viral envelope that surrounds the core. The viral-encoded replication
enzymes, including reverse transcriptase and integrase, are located within the viral core.
Fig. 2. The retrovirus replication cycle. Following recognition of a specific cellular receptor by the viral
envelope glycoprotein and adsorption of the virion to the cell surface, fusion of viral and cellular membranes
results in release of the viral core into the cell cytoplasm (usually at the cell surface, but for some retroviruses
this occurs following endocytosis of virions). The viral RNA is uncoated and reverse transcribed into a
double-stranded DNA copy. Following breakdown of the nuclear envelope, the DNA copy is integrated into
a chromosome where it resides as the provirus. Transcription of the provirus results in production of copies
of viral genomic RNA, which can either be translated to produce viral proteins or packaged by viral proteins
to form new core particles. Cores form and mature while budding from the cell surface, during which time
they obtain their envelopes consisting of cellular membrane and viral envelope glycoproteins (modified
from ref. 1).
viral RNA genome facilitate a strand transfer of the reverse transcriptase and nascent minus
strand DNA that is necessary for DNA synthesis to continue.9 Utilization of the polypurine
tract (ppt) located near the 3 end of the genome to initiate synthesis of the DNA plus strand
followed by a second strand-switching event results in the completion of reverse transcription
and the existence of a full-length double-stranded DNA copy of the viral genome containing
LTRs at each end. This genome is integrated, in an apparent random fashion, into a chromosome of the host cell where it resides as the provirus. The integration reaction11,12,13 is mediated by the viral integrase protein; the termini of the DNA genome are processed (two nucleotides on each end are removed), there is a break in and a short duplication made of the cellular
DNA sequence, the viral DNA is inserted, and DNA repair enzymes complete the integration
process. Because oncoretroviruses, unlike lentiviruses, lack a nuclear transport function to move
the proviral preintegration complex into an intact nucleus, the integration process occurs only
in cells where the nuclear envelope has broken down as a part of the mitotic process.14
Once integration of the viral genome is complete, the provirus is maintained in the cell
just like any cellular gene. A cellular RNA polymerase uses the viral promoter/enhancer in the
5 LTR to initiate transcription of proviral DNA. RNA transcripts are polyadenylated and
transported from the nucleus for translation as any cellular mRNA would be. The viral Gag
and Pol proteins are translated from a full-length unspliced RNA that extends from the 5 LTR
to the 3 LTR. Usually, during translation of the retroviral RNA, translation terminates after
reading of the gag open reading frame by the ribosomal complexes, resulting in significant Gag
production. However, about 5% of the time there is production of a Gag-Pol fusion polyprotein
precursor [some studies have indicated that the stoichiometry of Gag and Pol production appears to be important for efficient and correct virion assembly15,16]. The mechanism of production of the Gag-Pol precursor varies among the different retroviruses: in some retroviruses,
production of Gag-Pol during translation is a result of a ribosomal frameshift event that puts
the pol gene into the same reading frame as the gag gene;17 in other retroviruses, Gag-Pol
production results from a readthrough event mediated by a suppressor tRNA that prevents
termination of translation at the end of the gag gene.18
The envelope protein is expressed from a spliced message. This protein precursor undergoes post-translational modification including extensive glycosylation and processing by a cellular protease to generate the two TM and SU glycoprotein subunits that make up the functional Env protein.
Full-length transcripts of the viral genome, in addition to being translated into viral proteins, also can be incorporated into newly forming viral particles.19 This packaging (or
encapsidation) of the viral genome into newly forming capsids is mediated by an RNA packaging (encapsidation) signal located on full-length viral transcripts. This predominate packaging signal, sometimes referred to as or E, is generally located near the 5 end of the
genome between the splice donor and the gag start codon (Fig. 3). Because it is typically located
within an intron, the packaging signal is removed during splicing, insuring that only fulllength viral transcripts and not spliced transcripts (containing only env) are incorporated into
progeny virions.
Although the locations of retroviral packaging sequences are generally thought to be as
described above, further study of the packaging signals of various retroviruses has revealed that
the actual situation is, not surprisingly, more complex.20,21 In some cases, sequences upstream
of the splice donor have been shown to be important in RNA packaging. In other cases, sequences extending past the gag start codon have been shown to increase efficiency of RNA
encapsidation (these extended packaging signals are oftentimes referred to as + or E+).
Still, in other cases, sequences distant from the major packaging signal have been suggested to
affect encapsidation of viral RNA. It is generally believed that the secondary structure of the
RNA within the packaging signal, and not the primary RNA sequence itself, plays the more
important role in RNA encapsidation.
The process and relative timing of many events of virus particle assembly are not understood completely. Newly synthesized Gag proteins recognize RNA that is to be packaged into
virions and incorporate the RNA into capsids formed of multimers of Gag molecules. Recognition of the viral encapsidation sequence is thought to be mediated primarily by the NC
portion of the Gag protein precursor. Viral core formation is thought to be driven by intermolecular Gag-Gag interactions, with the Pol protein being incorporated into the forming virion
via Gag molecules interacting with the Gag portion of the Gag-Pol precursor. Viral core assembly and processing of the Gag and Gag-Pol protein precursors to form mature, infectious virions appears to occur during and just after budding of virus particles from the cell.22 The viralencoded protease (PR, encoded by pol) self-cleaves the Gag-Pol precursor and also further
processes Gag into the MA, CA, and NC proteins and Pol into the RT and IN proteins. The
Fig. 3. Retroviral vector cis-acting elements. In this example of a typical retroviral vector, the viral genes
have been removed and replaced with a foreign gene of interest. The viral sequences that remain as part of
the vector construct are necessary in cis during various steps of the retroviral replication cycle. These
sequences are necessary for vector production and for successful reverse transcription and integration of the
vector genome, followed by expression of the foreign gene. Although in the example shown the foreign gene
is expressed directly from the LTR, other strategies for expressing foreign genes exist and are illustrated in
Figure 4. att, attachment site; pbs, primer binding site; ppt, polypurine tract. Modified from ref. 1.
virion envelope, made up of cellular membrane and viral envelope glycoproteins, is obtained
during budding of the virus from the cell.
virus. This requirement can be satisfied by expression of the gag, pol, and env genes, removed
from the parental virus during vector construction, in cells that also express the vector construct. In this way, Gag, Pol, and Env can be provided in trans but the gag, pol, and env genes
will not be carried along with the vector when it is harvested and used to infect target cells.
Various strategies24 for expression of vector constructs and viral genes and for production of
vector virus are discussed below.
Vector virus that is produced by this trans-complementation can then be used to transduce (infect and express a foreign gene in) target cells. For experiments using vectors to study a
single round of viral replication, the foreign gene is generally some type of marker gene that can
be used to screen for transduction and to quantify the vector virus that is produced; typical
markers used to titer vector produced include lacz or genes encoding GFP or a molecule conferring antibiotic resistance.
Design of Vectors
Once the basic vector backbone has been constructed (Fig. 3), there are several different
ways in which to express a foreign gene (Fig. 4). In the simplest case, the foreign gene is expressed directly from the promoter located in the 5 LTR of the vector construct. Two or more
genes can be expressed from the LTR if the second gene is expressed from a spliced message or
if the second gene is translated using an internal ribosome entry site (IRES) (ref. 25). Although
using the vector LTR to drive expression of the foreign gene(s) is useful in a number of settings,
at times other strategies are used in order to attempt to control or to increase the level of gene
expression. By designing vectors that contain internal, heterologous promoters, oftentimes foreign gene expression can be increased greatly compared to levels that could be obtained using
the promoter in the vector LTR (this is attributable to low LTR promoter activity in the target
cell, which typically is not the natural target cell of the parental virus). Frequently, the heterologous promoter is another viral promoter, such as cytomegalovirus (CMV), or it may be a
tissue-specific promoter.
Of course, the years of experience with retroviral vector development have made it clear
that the design of vectors to deliver and express foreign genes in cells is often not as simple as
one might think from the description above. For example, sustained expression of the foreign
genes has been a great problem that can have many possible etiologies. For instance, although
use of a heterologous promoter can increase foreign gene expression, addition of an additional
promoter on the vector sometimes can actually decrease gene expression. This phenomenon,
termed promoter interference, is incompletely understood and not always predictable: often
it can be affected by the precise or relative location of the promoters or even by the foreign gene
itself.26,27 Efforts to overcome promoter interference have included attempts to express the
foreign gene from a heterologous promoter/foreign gene cassette placed on the vector in an
anti-sense orientation relative to the LTRs. This strategy has had mixed success, probably since
the same number of promoters are still present on the vector and possibly because the production of anti-sense transcripts may interfere with vector production or gene expression.
The development of self-inactivating (SIN) vectors may decrease the problem of promoter interference and also offers a potential safety advantage over traditional retroviral vectors. Sin vectors are named as such because they are engineered to generate, following reverse
transcription of the vector RNA into the DNA form, a defective, and thus inactive, promoter
in the 5 LTR. This is accomplished by engineering constructs that have a defective u3 region at
the 3 end of the viral RNA form of the genome (this defective u3 is duplicated as a defective
U3 of the 5 LTR during reverse transcription; see above and Chapter 2). In this scenario, there
would be no active promoter in the proviral 5 LTR to interfere with internal, heterologous
promoters. It also, in theory, offers additional safety advantages in that it would be more difficult to re-generate a wild-type parental retrovirus via recombination and also it may decrease
Fig. 4. Vector design strategies for expression of foreign genes. Examples of various strategies for expression
of foreign genes delivered by retroviral vectors are illustrated. Other combinations of the strategies shown
here also can be used. A. The foreign gene is expressed directly from the promoter located in the vector LTR.
B. Expression of two or more foreign genes might be expressed using the promoter in the vector LTR by
utilizing a spliced message to express the second gene. C. A foreign gene can be expressed from a heterologous
promoter located in the middle of the vector; sometimes this promoter/foreign gene cassette is placed in the
anti-sense orientation relative to the vector LTR. D. An internal ribosome entry site (IRES) can be utilized
to express a second foreign gene. LTR, long terminal repeat; S.D., splice donor; S.A., splice acceptor; Pr,
promoter. Arrows indicate the location and direction of transcription initiation.
the chances of insertional mutagenesis by a promoter insertion mechanism after the provirus is
integrated into the cells genome.
package vector RNA, and vector virus that is released from these transfected cells can be harvested for transduction of target cells. Because the helper packaging plasmids do not contain
the cis-acting sequences necessary for propagation (see above), they will not be packaged or
transferred to the target cells; therefore, there will be only a single round of vector replication
and the foreign gene can be introduced into target cells in a relatively predictable manner.
In order to decrease the chances of the re-generation of wild-type virus (replication-competent retrovirus, RCR) during vector production via recombination between vector and packaging constructs, use of a split genome approach to expressing viral proteins generally has
been adopted. In such an approach, the vector is expressed on one construct, the gag and pol
genes on another, and the env gene on yet another. Separate expression of the env gene also
enables the use of Env from heterologus viruses in place of the native Env. The use of Env from
heterologous viruses, termed pseudotyping, is extremely valuable because it allows the cellular
tropism of the vector virus to be different from that of the parental virus from which the vector
was derived. In addition, different envelope proteins have different physical properties that can
be an advantage during vector preparation.28,29 The split genome approach decreases the frequency of RCR generation, given that multiple recombination events would need to take place.
However, it does not reduce it to zero, especially when using transient transfection of cells, an
inherently recombinagenic procedure, to produce vector virus. For this reason, packaging cell
lines often are used to generate vector virus.
Packaging cells are cell lines that have been engineered to stably maintain viral genes and
to express viral proteins.30 The viral genes are expressed from heterologous promoters and there
are no cis-acting sequences associated with the viral genes, just as when packaging plasmids are
used in transient transfections as described above; usually the split genome approach is used to
express gag, pol, and env. When a vector construct is introduced into the packaging cells, usually by transfection, vector virus is produced and can be harvested (Fig. 5). Generally, the use of
packaging cells was thought to be preferential to the use of a transient transfection protocol for
vector production: packaging cells can be well characterized and it was thought that the chances
of RCR formation through recombination was less than during a transient co-transfection of
vector and packaging plamsids. The use of packaging cells was generally a more direct way to
produce vector virus and yielded higher vector titers than those obtained from transient transfections; however, improvements in protocols for vector production have made this factor less
of a concern.
One final word on vector production and design of packaging systems: the biggest safety
concern31 during vector production is that RCR might inadvertently be generated.32 Therefore, improvements designed to reduce the amount of sequence homology among constructs
used in vector and packaging systems has remained a priority. Still, all vector preparations used
need to be extensively tested for the presence of RCR.
Summary
Vector systems based on oncoretroviruses have been important tools for understanding
basic retroviral replication and for applied studies involving gene transfer. Development of
these vector systems enabled detailed study of the basic biology of the parent retrovirus from
which they were derived and vice versa, enabling advances in vector systems and gene transfer
technology to be made. Use of retroviral vectors for gene transfer has been limited for a number
of years by several problems including low vector titers, inability to achieve sustained foreign
gene expression in target cells, inability to target vector virus to transduce the desired cell type,
the theoretical possibility of insertional mutagenesis by the vector upon integration, and the
possibility of the re-generation wild-type virus during vector production. Over time, advances
in the understanding of retroviral vector design and gene transfer have occurred and progress
has been made in overcoming what had been identified as system limitations.
10
Fig. 5. Vector production using a packaging cell line. Packaging cells are cell lines that have been engineered
to stably express viral proteins from heterologous promoters; these viral protein-coding sequences lack cisacting sequences that are necessary for propagation and are therefore not transmitted to other cells. Following introduction of a vector into the packaging cell, usually by transfection, vector RNA is produced and
packaged into vector virus particles that are then released from the packaging cell. These vector virus particles
can be harvested and used to infect fresh target cells. Following reverse transcription and integration of the
vector genome, the foreign gene is expressed in the target cells. Because viral proteins are not produced in
the target cells, further vector production and propagation does not occur (1).
Recently, vector systems derived from several different lentiviruses, which could offer some
advantages over oncoretroviral vector systems, have been under development. At least some of
these systems are anticipated to have use in future clinical gene transfer applications. Certainly,
the immense experience obtained with oncoretroviral vector systems and the basic tenets that
guided their development has benefited and will continue to benefit the development of lentiviral
vector systems which, because of their more complex genome and their potential to be human
pathogens, raise technical, practical, and ethical issues that must be addressed.
Acknowledgements
I thank Kathleen Boris-Lawrie of the Ohio State University for comments on the manuscript and Scott Buchschacher for preparation of figures.
References
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Philadelphia: Lippincott, Williams & Wilkins, 2000:149-158.
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17. Jacks T, Power FR, Masiarz PA et al. Characterization of ribosomal frameshifting in HIV-1 gagpol expression. Nature 1988; 331:280-283.
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19. Butsch M, Boris-Lawrie K. Translation is not required to generate virion precursor RNA in human
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20. McBride MS, Panganiban AT. The human immunodeficiency virus type 1 encapsidation site is a
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12
CHAPTER 2
HIV-1 Replication
Eric O. Freed
Introduction
n general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles
that of other retroviruses.1 There are, however, a number of unique aspects of HIV
replication; for example, the HIVs and SIVs target receptors and coreceptors distinct from
those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory
proteins not encoded by the genomes of the prototypical simple retroviruses. Of particular
interest from the gene therapy perspective, lentiviruses possess the ability to productively infect
some types of non-dividing cells. This chapter, while reiterating certain points discussed in
Chapter 1, will attempt to focus on issues unique to HIV-1 replication.
The HIV-1 genome encodes the major structural and non-structural proteins common to
all replication-competent retroviruses (Fig. 1, and Chapter 1). From the 5'- to 3'-ends of the
genome are found the gag (for group-specific antigen), pol (for polymerase), and env (for envelope glycoprotein) genes. The gag gene encodes a polyprotein precursor whose name, Pr55Gag ,
is based on its molecular weight. Pr55Gag is cleaved by the viral protease (PR) to the mature
Gag proteins matrix (also known as MA or p17), capsid (CA or p24), nucleocapsid (NC or
p7), and p6. Two spacer peptides, p2 and p1, are also generated upon Pr55Gag processing. The
pol-encoded enzymes are initially synthesized as part of a large polyprotein precursor, Pr160GagPol,
whose synthesis results from a rare frameshifting event during Pr55Gag translation. The individual pol-encoded enzymes, PR, reverse transcriptase (RT), and integrase (IN), are cleaved
from Pr160GagPol by the viral PR.
The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor (Fig. 1).
Unlike the Gag and Pol precursors, which are cleaved by the viral PR, the Env precursor,
known as gp160, is processed by a cellular protease during Env trafficking to the cell surface.
gp160 processing results in the generation of the surface (SU) Env glycoprotein gp120 and the
transmembrane (TM) glycoprotein gp41. gp120 contains the determinants that interact with
receptor and coreceptor, while gp41 not only anchors the gp120/gp41 complex in the membrane (Fig. 2), but also contains domains that are critical for catalyzing the membrane fusion
reaction between viral and host lipid bilayers during virus entry. Comparison of env sequences
from a large number of virus isolates revealed that gp120 is organized into five conserved
regions (C1-C5) and five highly variable domains (V1-V5). The variable regions tend to be
located in disulfide-linked loops. gp41 is composed of three major domains: the ectodomain
(which contains determinants essential for membrane fusion), the transmembrane anchor sequence, and the cytoplasmic tail.
In addition to the gag, pol, and env genes, HIV-1 also encodes a number of regulatory and
accessory proteins. Tat is critical for transcription from the HIV-1 LTR and Rev plays a major
Lentiviral Vector Systems for Gene Transfer, edited by Gary L. Buchschacher, Jr.
2002 Eurekah.com.
HIV-1 Replication
13
Fig. 1. Organization of the HIV-1 genome. The relative locations of the HIV-1 open reading frames gag,
pol, env, vif, vpr, vpu, nef, tat, and rev are indicated. The 5' and 3' LTRs are shown, with U3, R, and U5 regions
noted. The indicates the position of the RNA packaging signal. The major Gag domains (MA, CA, NC,
p6) and the Gag spacer peptides (p2 and p1) are shown under the gag gene. The site of Gag N-terminal
myristylation is denoted as myr. Under the pol gene are indicated the PR, RT (p66 and p51 subdomains),
and IN coding regions. The SU and TM Env glycoproteins (gp120 and gp41, respectively) are enlarged to
show the position in gp120 of the major conserved (C1-C5) and variable (V1-V5) regions and in gp41 the
location of the fusion peptide, the N- and C-helices, membrane-spanning domain, and the cytoplasmic tail.
role in the transport of viral RNAs from the nucleus to the cytoplasm. Vpu, Vif, Vpr and Nef
have been termed accessory or auxiliary proteins to reflect the fact that they are not uniformly required for virus replication. The functions of these very interesting proteins will be
discussed in more detail at the end of this chapter.
HIV replication proceeds in a series of events that can be divided into two overall phases:
early and late (Fig. 3).1 Although some events occur in a concerted or simultaneous fashion, the replication cycle can be viewed most simply as proceeding in an ordered, step-wise
manner. In this chapter, each step in virus replication will be considered; additional information can be obtained from the more detailed reviews and primary references that are cited.
14
Fig. 2. Schematic representation of a mature HIV-1 particle. Positions of the major viral proteins, the lipid
bilayer, and the genomic RNA are indicated. Modified from Freed, 1998 (ref. 22)
crystal structure of the gp120 core reveals two major domains, the inner and outer domains, connected by a bridging sheet.3 One of the interesting features of the gp120 structure
evident from this work is that the CD4 binding site in gp120 is deeply recessed and flanked by
heavily glycosylated variable regions. A number of direct gp120/CD4 contacts are resolved in this
structure.
As a consequence of the high affinity interaction between gp120 and CD4, these two
molecules associate during their transport to the surface of infected cells in the late phase of the
replication cycle.4 This intracellular Env/CD4 interaction leads to the downmodulation of
CD4 from the cell surface, rendering infected cells partially resistant to further infection by
HIV Env-bearing viruses. This Env-mediated superinfection interference is not operative
against virions bearing heterologous Env glycoproteins [e.g., those of amphotropic murine
leukemia virus (A-MuLV) or the vesicular stomatitis virus G glycoprotein (VSV-G)] as such
pseudotyped viruses bind and enter the target cell in a CD4-independent manner.
Coreceptor Interactions
After the identification of CD4 as the major HIV receptor, it was soon appreciated that
CD4 was not sufficient for HIV Env-mediated membrane fusion and virus entry. This conclusion was based in part on the observation that primary virus isolates from infected individuals
display variable tropism for CD4+ cells. Certain isolates, referred to as macrophage-tropic (or
M-tropic) replicate efficiently in primary macrophage cultures, whereas other isolates, referred to as T-cell-line tropic (T- or TCL-tropic) cannot productively infect macrophages
but replicate to high levels in T-cell lines. Both M- and TCL-tropic isolates replicate in activated peripheral blood mononuclear cells (PBMC). A decade-long search ultimately identified
members of the G protein-coupled receptor superfamily of seven-transmembrane domain proteins as coreceptors for HIV entry. 5,6 These molecules serve as receptors for the and
HIV-1 Replication
15
Fig. 3. Schematic representation of the HIV-1 life cycle. The major steps in the early and late stages of the
replication cycle (described in detail in the text) are indicated.
16
Fig. 4. Steps leading to membrane fusion induced by HIV-1 Env. Panel 1. The gp120/gp41 complex in its
resting configuration prior to interaction with CD4 and coreceptor. Panel 2. gp120 binds CD4 and
interacts with coreceptor, leading to conformational changes in both gp120 and gp41. Panel 3. The gp41
ectodomain adopts a hypothetical extended conformation; the fusion peptide at the N-terminus of gp41
inserts directly into the target cell lipid bilayer. Panel 4. The N- and C-helices of the gp41 ectodomain fold
into a highly stable six-helix bundle, bringing the membranes in apposition and allowing membrane fusion
to occur. Modified from Freed and Martin, 2001 (see ref. 1).
completely resistant to HIV infection.9-11 These observations suggest that coreceptors might
be effective targets for antiviral therapy.
Membrane Fusion
The membrane fusion reaction that takes place between the lipid bilayers of the viral
envelope and the host cell plasma membrane enables the viral core to gain access to the cytoplasm and is thus central to the infection process. As mentioned above, membrane fusion is
catalyzed by the gp120/gp41 Env glycoprotein complex (Fig. 4). The role of gp120 is primarily
to bind the target cell, interact with coreceptor, and induce conformational changes in gp41
that allow it to directly promote the fusion event. A number of domains in both gp120 and
gp41 participate in CD4/coreceptor binding or the membrane fusion process itself. At the
heart of the fusion reaction is the ectodomain of gp41; this region contains a highly hydrophobic N-terminus (the so-called fusion peptide) and two heptad repeat motifs, referred to as the
N-helix and the C-helix (Figs. 1 and 4). Structural analyses of these two helical sequences, in
the context of a gp41 ectodomain trimer, indicate that they pack in an antiparallel fashion to
generate a six-helix bundle 12,13 (Fig. 4). Mutations within, or peptides derived from, these
heptad repeats potently inhibit membrane fusion. It appears that the N- and C-helices undergo
HIV-1 Replication
17
rearrangements following CD4/coreceptor binding that enable the N-terminal fusion peptide
to insert directly into the target membrane. In many respects, this spring-loaded mechanism
of membrane fusion closely parallels the fusion model proposed for influenza HA2.14
Post-Entry Events
Following the fusion between the envelope of the incoming virus particle and the host cell
plasma membrane, the viral core enters the host cytoplasm. The events that follow remain the
least well understood part of the virus life cycle. In particular, the process of uncoating,
during which the core (defined as the structure that remains after the lipid bilayer is stripped
away) is converted to a complex referred to as the reverse transcription complex (RTC) and
then the preintegration complex (PIC). During these steps, CA appears to be lost while at least
some MA, NC, the pol-encoded enzymes RT and IN, and the accessory protein Vpr, remain
associated. Because of the very high ratio of physical particles to infectious units in HIV-1
preparations (approximately 1000:1), these uncoating steps are very difficult to study; the vast
majority of viral protein that enters the target cell does not lead to a productive infection.
However, successful virus entry can be followed by monitoring reverse transcription and, ultimately, integration.
Reverse Transcription
One of the defining features of retroviruses is their ability to convert their RNA genomes
into double-stranded DNA early post-infection. This reaction is catalyzed by the RT enzyme.15
In the case of HIV-1, RT is a heterodimer of two subunits, one of which is 66 kDa (p66), the
other 51 kDa (p51). These two subunits are both derived from the same region of the Pr160GagPol
precursor protein; p51 is formed when the C-terminal, 15kDa RNaseH domain of p66 is
removed by PR. A number of groups have crystallized the RT holoenzyme in various contexts,
leaving us with a very well-defined structure. Interestingly, the p66 and p51 domains, while
largely overlapping in protein sequence, adopt quite different conformations. The p66 subunit
can be visualized as a right hand, with the polymerase active site within the palm, and a deep
template-binding cleft formed by the palm, fingers and thumb subdomains.16 In this representation, the active site is located in the palm.
Reverse transcription proceeds in a series of steps that utilize several cis-acting elements in
the viral genome.15 With one interesting distinction, mentioned below, reverse transcription of
the HIV-1 genome occurs by a process that is fundamentally similar to that used by other
retroviruses. The steps involved in the conversion of the RNA genome to DNA are described
briefly below, and are depicted in Fig. 5.
1. Reverse transcription is initiated using as a primer a molecule of tRNA that is bound to the
primer binding site (pbs). DNA synthesis proceeds to the 5' end of the RNA molecule,
generating a DNA/RNA hybrid.
2. The RNA portion of this hybrid is degraded by the RNaseH activity that is an inherent part
of the RT holoenzyme, generating a DNA fragment known as the minus-strand strong stop
DNA.
3. By using short regions of homology (the so-called R regions), the minus-strand strongstop DNA jumps from the 5' to the 3' end of the genome. This step is referred to as the
first strand transfer.
4. Minus-strand synthesis occurs, using the 3' end of the minus-strand strong stop DNA as a
primer.
5. Plus-strand synthesis occurs, using as primers fragments of RNA remaining from minusstrand synthesis. The primary site of priming for retroviruses takes place at a purine-rich
sequence known as the polypurine tract (PPT). For HIV, priming also occurs efficiently
18
Fig. 5. Reverse transcription. Thin lines represent RNA; DNA is indicated as a thick line. Cis-acting
elements, defined in the text, are shown. The tRNA primer is shown bound to the pbs.
from another site, known as the central PPT. The implications of priming at the central PPT
will be discussed further in the context of nuclear import.
6. The tRNA bound to the pbs is removed by RNaseH, thereby allowing second-strand transfer to take place.
HIV-1 Replication
19
7. Plus-strand synthesis proceeds to the end of the minus strand. For HIV, an additional termination site, referred to as the central termination signal (CTS), is located near the center of the
genome. Since the CTS is 3' of the central PPT, approximately 100 nucleotides of plusstrand DNA is displaced, resulting in the formation of a DNA flap. It has been reported
that this central flap plays a crucial role in the import of the viral PIC to the nucleus.17
HIV, and other retroviruses, package two single stranded copies of their RNA genome per
virion. Because reverse transcription involves jumps from one template to another, the RT/
template interaction is of a relatively low affinity.18 As a consequence, frequent template switches
can occur; if the two RNA molecules in a particular virion are not genetically identical, this
template switching will result in the generation of a novel recombinant DNA genome containing sequences derived from both parental RNAs.19 The high frequency of genetic recombination, together with the high mutation rate of HIV-1 RT (3 X 10-5 per cycle of replication20)
results in HIV populations being highly heterogeneous in sequence (forming so-called
quasispecies). As a consequence, HIV is able to rapidly evade the host immune response and
develop resistance to antiviral drugs.
The RT enzyme was an early target in the development of antiviral drugs. Two general
classes of RT inhibitors have been developed: the nucleoside analogs [e.g., AZT (zidovudine),
ddI (didanosine), ddC (zalcitabine), and 3TC (lamivudine)] and the nonnucleoside inhibitors
(e.g., nevirapine and delavirdine). Because of the above-mentioned high mutation rate of RT,
resistance to these compounds emerges rapidly; however, potent treatment regimens result
from the combination of RT and PR inhibitors.
Nuclear Import
During reverse transcription, the viral genome (RNA, then DNA) remains associated
with the high molecular weight RTC. The viral DNA is eventually transported to the nucleus
as part of the PIC. As mentioned above, the composition of the PIC, and the mechanism by
which the PIC translocates to the nucleus, have been the subject of some debate. There appears
to be widespread agreement that CA is not part of the PIC, but that at least some MA is
retained. The pol-encoded enzyme IN, which of course must be present in the nucleus at the
time of integration (see below), is also part of the PIC. The accessory protein Vpr and, in some
studies, the Gag NC protein, are also detected in this complex. Although it was originally
proposed that MA was the primary determinant of PIC nuclear import,21 certain key pieces of
data supporting this model were not reproduced in subsequent studies.22 It is currently believed that Vpr plays a role in nuclear import, and some studies suggest a role for IN.23 Most
recently, as alluded to above, the central DNA flap that is a product of lentiviral reverse transcription has been implicated in PIC nuclear import.17 It must be emphasized that our understanding of this process is incomplete and that further studies will be required to elucidate the
role of viral, and, presumably, cellular factors in the translocation of the PIC to the nucleus.
This topic will be discussed in much greater detail in Chapter 3.
Integration
Following nuclear import of the viral PIC, the 32 kDa IN protein catalyzes the insertion
of the linear, double-stranded viral DNA into the host cell chromosome.24 The integrated
DNA, referred to as the provirus, behaves essentially as a cellular gene. Integration is an
essential step in retrovirus replication, as IN mutants generally fail to establish spreading infections. As with reverse transcription, integration proceeds via a well-defined series of steps that
are quite similar among all retroviruses. 1) The integration process begins when IN clips off
several nucleotides from the 3 termini of both strands of linear viral DNA. This reaction,
known as 3'-end processing, generates a molecule of double-stranded DNA with 3-recessed
ends. 2) In the nucleus, IN makes a staggered cleavage in the cellular target DNA. The 3
20
recessed ends of viral DNA formed in the 3'-end processing reaction are joined to the ends of
the cleaved cellular DNA. This reaction is known as strand transfer. The integration process is
completed when cellular repair enzymes fill in the gaps between the integrated viral DNA and
the host target DNA.
The IN enzyme is composed of three distinct domains: an N-terminal domain that contains a zinc-finger, a central core sequence, and a C-terminal domain. The core contains the
active site of the enzyme and bears marked structural similarity to other polynucleotidyl transferases (e.g., the bacteriophage MuA transposase and RNAse H). Three highly conserved residues, Asp64, Asp116 and Glu152 (the so-called D,D-35-E motif ) are critical to IN function. It is
well established that IN functions as a multimer; however, it is still not clear how many molecules of IN make up the functional holoenzyme. Recent studies suggest that not only is the
viral DNA present in a large macromolecular PIC, but that the ends of the viral DNA are also
organized into a multi-component complex composed of both viral and cellular proteins. These
complexes at the viral DNA ends are referred to as intasomes.25,26
Much of what we know about retroviral integration derives from the study of in vitro
integration reactions using purified IN. Despite the utility of this simplified system, the major
product of such reactions using HIV-1 IN is a single end of viral DNA joined to one strand of
the target, rather than the product observed in vivo in which both ends of the viral DNA are
integrated. This observation highlights the importance of additional viral and perhaps cellular
proteins in carrying out the integration reaction. Indeed, when PICs are purified from infected
cells they are able to direct authentic and complete integration reactions in vitro. Such reactions provide a powerful tool to screen for compounds that block integration in vivo.27
Gene Expression
Following integration into the host chromosome, the integrated provirus serves as the
template for the synthesis of the viral RNAs that ultimately encode the full complement of
structural, regulatory, and accessory proteins used to direct virus replication. In this section, we
will consider the transcription of the viral RNAs, and the role of the Tat protein in RNA
synthesis, and will summarize the role of Rev in the export of viral RNAs to the cytoplasm.
Transcription/Tat Function
The HIV-1 LTR serves as the site of transcriptional initiation and harbors cis-acting elements required for RNA synthesis1 (Fig. 6). The LTR is composed of three regions: U3 (for
unique, 3' end), R (for repeated) and U5 (for unique, 5' end). Transcription initiates at the U3/
R junction. U3 contains a variety of elements that direct the binding of RNA polymerase II
(pol II) to the DNA template. A TATA element, to which transcription factor IID (TFIID)
binds, is located approximately 25 nucleotides upstream of the transcription start site. Located
5' of the TATA box are three Sp1 and two NF-B binding sites. Although mutational analyses
of the HIV-1 LTR reveal that the Sp1 and NF-B sites are variably important, depending upon
the cell type, removal of all Sp1 and NF-B sites abolishes virus replication.28 Upstream of the
NF-B sites is a domain, sometimes referred to as the modulatory region, which contains
binding sites for several additional transcriptional factors, including LEF, Ets, and USF.
The basal transcriptional activity from the HIV LTR is very low; RNA synthesis is greatly
increased (by more than two logs) when the transcriptional transactivator protein Tat is
present.29,30 Tat is a 101 amino acid protein encoded by a two-exon RNA; a smaller (72 amino
acid) one-exon Tat is encoded by some isolates. Tat contains several distinct functional domains: an activation domain, which lies within the N-terminal 48 residues of the protein and
which itself is comprised of an acidic domain, a Cys-rich region, and a hydrophobic core element; a highly basic RNA binding domain; and an overlapping nuclear localization signal.
HIV-1 Replication
21
Fig. 6. Schematic representation of the HIV-1 LTR. The position of binding sites for host factors (LBP-1,
NFB, LEF, Ets, USF-1, and NFAT-1) are shown at and 5' of the transcription start site. The TAR stem/
loop structure, with bulge, is represented at the 5' end of a nascent mRNA.
Much effort over the past decade has been focused on understanding the mechanism by
which Tat transactivates LTR-driven gene expression. Several salient features are emphasized
below: 1) Tat acts upon an RNA element, known as the transactivation response region (TAR).31
TAR, which is present at the 5' end of all viral RNAs, consists of a base-paired stem, a small
(trinucleotide) non-base-paired bulge, and a 6-nucleotide G-rich loop (Fig. 6). 2) Tat appears
to bind to the TAR bulge. 3) It was observed a number of years ago that Tat functions poorly in
rodent cells, suggesting that cellular factors play an important role in Tat activity. This prediction was borne out recently with the identification of a cellular protein that interacts, via its
activation domain, with Tat.32 The protein in question is cyclin T1 (cycT1), which forms a
heterodimer with a member of the cyclin-dependent kinase family (CDK9). The cycT1/CDK9
heterodimer is itself part of a large protein complex related to the Drosophila positive-transcriptional elongation factor b (P-TEF-b). Tat recruits the human P-TEFb complex to TAR,
resulting in the phosphorylation of the C-terminal domain (CTD) of RNA Pol II and a dramatic stimulation of transcriptional processivity.
22
of interacting with the cellular nuclear export machinery. As a consequence, the RRE-containing RNA is transported, in an unspliced or partially spliced form, to the cytoplasm. Rev then
shuttles back to the nucleus, using its nuclear localization signal. An additional level of complexity is provided by the presence in HIV-1 RNAs of cis-acting elements, located within gag,
pol, and env, whose presence inhibits RNA utilization in the absence of Rev.35,36 These elements likely bind cellular proteins that retain RNAs containing the elements in the nucleus.
Rev counteracts this effect, allowing efficient RNA export to proceed.
It is probable that the more simple retroviruses contain in their RNAs RRE-like sequences that interact with cellular Rev-like proteins to facilitate their transport to the cytoplasm. Indeed, such an element, referred to as the constitutive transport element (CTE) has
been identified in the genome of Mason-Pfizer Monkey virus.37 By introducing the CTE in
HIV-1 RNAs, the requirement for Rev and the RRE can be bypassed.
HIV-1 Replication
23
Fig. 7. Functional domains of the HIV-1 Gag domains MA (A), CA (B), NC (C), and p6 (D). Details are
provided in the text. Adapted from Freed, 1998 (ref. 22).
plays a role in this process. Mutations within MA can re-route assembly to intracellular compartments (e.g., the ER or Golgi),41,42 and substitution of MA with a heterologous membrane
binding signal results in promiscuous targeting of virus assembly to both plasma and intracellular membranes.
RNA Encapsidation
As discussed above, each retrovirus particle contains two single-stranded copies of genomic RNA. The cis-acting sequence that directs RNA encapsidation, known variously as the
packaging signal, encapsidation element, or -site, is typically located in the region of the
RNA 5' of the gag initiation codon.43 In the case of HIV-1, the packaging signal appears to be
more dispersed than in the murine and avian retroviruses and a larger region of the genome is
required for efficient RNA packaging. The HIV-1 packaging signal is composed primarily of
four stem-loop structures, referred to as SL1-SL4, although sequences outside this region contribute to efficient RNA encapsidation. The secondary structure of this packaging signal, rather
24
than the actual nucleotide sequence itself, seems to be important in conferring RNA
encapsidation specificity. RNAs that lack the packaging signal are not efficiently encapsidated
into virus particles. Retroviral RNAs are linked together at a sequence near the 5' end of the
genome known as the dimer initiation signal (DIS). It is currently unclear to what extent RNA
dimerization is required for efficient encapsidation.
The specific encapsidation of retroviral RNAs into virus particles is mediated by interactions between the packaging signal and the NC domain of Gag. Nearly all retroviral NC proteins contain one or two zinc finger motifs, each of which coordinates a zinc ion. HIV-1 NC
contains two zinc-finger motifs (Fig. 7) of the CCHC type (Cys-X2-Cys-X4-His-X4-Cys;
where X denotes a variable amino acid). The zinc fingers of HIV-1 NC are flanked by highly
basic sequences. NC displays both non-specific nucleic acid binding properties and specific
genomic RNA binding; in general, the non-specific binding properties are conferred by the
basic residues, whereas the zinc finger motifs, in conjunction with the basic residues, contribute to the specificity of the NC/RNA interaction. It has been proposed that NC first interacts,
in a sequence-specific fashion, with the packaging signal (SL3 appears to be particularly important in this regard) then additional NC domains coat the viral RNA in a sequence-independent
manner. In addition to its function in RNA encapsidation, NC plays a role in a variety of
additional steps in the viral life cycle. In many cases these activities can be attributed to the
ability of NC to function as a nucleic acid chaperone.44 This property enables NC to refold
nucleic acid molecules to the most energetically favorable conformation.
In general, retroviruses are able to package their own RNAs genomes, but not those of
other retroviruses. The evaluation of chimeric viruses indicates, as mentioned above, that
encapsidation specificity is determined by NC. In some cases, non-reciprocal packaging can be
observed; for example, HIV-1 packages both HIV-1 and HIV-2 genomic RNAs, whereas HIV2 reportedly does not efficiently encapsidate HIV-1 RNA.45
Assembly
Once Gag has arrived at the plasma membrane, it must engage in Gag-Gag (as well as
Gag-lipid and Gag-RNA) interactions to enable the assembly of progeny virions to take place.
A number of approaches, including in vitro assembly reactions, mutational analyses, virion
incorporation assays, and yeast two-hybrid screens have suggested that several domains in Gag
are important in mediating Gag-Gag interactions.22 These domains primarily encompass a
region spanning the C-terminus of CA, the p2 spacer peptide, and the N-terminal portion of NC.
The HIV-1 CA folds into two distinct structural and functional domains: an N-terminal
core domain, and a C-terminal dimerization domain (Fig. 7). The C-terminal domain
harbors the so-called major homology region which contains residues conserved among many
retrovirus genera. Mutations within the C-terminal dimerization domain often cause defects in
virus assembly, suggesting the involvement of this region in Gag-Gag interactions. HIV-1 CA,
when expressed in vitro, can direct the assembly of tubular or spherical particles, again highlighting the role of CA in virus assembly. Mutations in NC, specifically within the highly basic
residues near the N-terminus of the protein, also induce defects in virus particle production.
This finding, together with the observation that CA-NC fusion proteins assemble in vitro
more efficiently than does CA alone, have suggested that NC also promotes Gag-Gag interactions. Interestingly, in vitro assembly of CA-NC fusion proteins requires the presence of nucleic
acid.46 This latter result suggests a model in which interactions between the Gag NC domain
and RNA allow Gag molecules to align and pack. This model is consistent with mutational
studies demonstrating that mutation in the basic residues within NC disrupt both Gag assembly and RNA binding.
HIV-1 Replication
25
Budding
The final step in the process of virus assembly and release involves the pinching off, or
budding, of the virus particle from the host cell plasma membrane. While it was felt initially
that budding was likely to be a spontaneous event, it has become clear that a wide range of
26
retroviruses (and at least several other enveloped viruses) encode specific sequences that promote particle release. These sequences are collectively referred to as late or L domains to
reflect their role late in virus assembly. Retroviruses encode their L domains at a variety of
positions in Gag; in the case of HIV-1, the L domain is present in p6 (Fig. 7). Deletion of p6,
or mutations within a highly conserved Pro-Thr/Ser-Ala-Pro (P-T/S-A-P) motif located near
the N-terminus of p6, markedly impair particle release.50,51 Examination of cells expressing
p6-mutant HIV-1 clones reveals the presence of large numbers of particles attached to the
plasma membrane by a thin tether, apparently unable to pinch off from the cell.
Although the mechanism by which L domains stimulate virus release remains to be elucidated, evidence is increasing that these domains function by interacting with host factors.
Most retroviral L domains contain the motif Pro-Pro-Pro-Tyr (P-P-P-Y), which is the consensus binding sequence for the so-called WW family of proteins. Indeed, direct interactions
between P-P-P-Y-type L domains and WW proteins have been detected in vitro.52 One of
these proteins, Nedd4, is a ubiquitin ligase. This latter observation, together with the finding
that proteosome inhibitors, which disrupt ubiquitination, impair both HIV and Rous sarcoma
virus release, suggests that L domains may function through the host ubiquitination pathway.53 How this would promote virus release remains an interesting question for future investigation.
Maturation
During or shortly after virus release from the plasma membrane, the viral PR cleaves the
Gag and GagPol polyprotein precursors to generate the mature Gag and Pol proteins (Figs. 1
and 3). Retroviral PRs are members of the family of aspartyl proteases. X-ray crystallography
indicates that retroviral PRs function as dimers, with the substrate-binding site located in a
cleft formed by two (identical) monomers.54
PR-mediated Gag and GagPol processing sets in motion a series of structural rearrangements that ultimately leads to virion maturation. PR cleaves each site with a differing efficiency; as a result, PR-mediated Gag and GagPol processing takes place as an ordered, stepwise cascade of cleavage reactions. The most visible outcome of HIV-1 maturation is that
virion morphology is converted from doughnut-shaped (containing an electron-lucent center)
to containing an electron-dense, conical core. Because of the degree and magnitude of protein
rearrangements that are presumed to occur during maturation, this process can be considered
as a second assembly (or re-assembly) reaction. Studies performed using cryo-EM techniques
have visualized unprocessed retroviral Gag monomers in immature virions as being aligned
likes spokes on a wheel, projecting inward from the membrane-associated MA domain at the
N-terminus to NC at the C-terminus.55 Following cleavage, CA forms a conical shell around
the RNA/protein complex within the core (Fig. 2). Numerous mutations have been reported,
many of which are located within the N-terminal domain of CA, that prevent the formation of
the normal conical core. Invariably, the failure of the virion to mature properly is associated
with a complete loss of infectivity; core condensation thus appears to be essential during an
early post-entry step of the replication cycle. Interestingly, core-like structures can assemble in
vitro from a CA-NC fusion protein in the presence of RNA. These cones closely resemble the
fullerenes formed by elemental carbon.56
During virus assembly, the N-terminal domain of CA binds, and ultimately packages into
virions, the host protein cyclophilin A.57,58 Mutations that disrupt CA-cyclophilin A binding, or
treatment of virus-infected cells with cyclosporin (which prevents cyclophilin A incorporation)
result in markedly impaired virus infectivity. The incorporation of cyclophilin A into virions is
HIV-1-specific, as neither HIV-2 nor SIV CA proteins bind this host factor. Although uncertainty
remains regarding the role that cyclophilin A plays in stimulating infectivity, it has been suggested
HIV-1 Replication
27
that this protein, which functions in the cell as a peptidyl-prolyl cis-trans isomerase, functions as a
chaperone during maturation to prevent unfavorable CA aggregation.59
The absolute requirement for PR-mediated virion maturation has been applied to the
treatment of HIV-infected individuals using inhibitors of PR. Although in infected patients,
the use of PR inhibitors alone rapidly leads to the generation of drug-resistant variants, when
combined with anti-RT inhibitors [(in so-called triple therapy or highly active antiretroviral
therapy (HAART)], long-lasting, clinically significant reductions in virus loads can be achieved.
Vpu
Vpu (for viral protein u) is an 81 amino acid integral membrane phosphoprotein that is
unique to HIV-1; with the exception of the highly HIV-1-related SIVcpz, it is not encoded by
the genomes of SIV or HIV-2 isolates. Vpu performs two major functions during HIV-1 replication: 1) it enhances the release of virus particles, and 2) promotes the degradation of CD4.
Mutational inactivation of Vpu reduces by several-fold the efficiency with which virions
are released from virus-expressing cells.61 This release function is independent of the p6 Gag L
domain (described above). Interestingly, although divergent retroviruses, such as MuLV and
visna, do not encode Vpu proteins, HIV-1 Vpu can stimulate their release as well.62 This
observation suggests that the enhancement of release occurs through a general pathway that
does not depend on the interaction of Vpu with specific viral factors.
HIV-1 has evolved several mechanisms to downregulate cell-surface expression of the major
receptor CD4. One mechanism involves intracellular trapping of CD4 by Env glycoproteins, a
second operates through Nef (see below) and the third is promoted by Vpu. CD4 degradation
by Vpu is mediated through the host ubiquitin/proteasome pathway.63 One outcome of Vpuinduced CD4 degradation is to liberate gp160 from Env/CD4 complexes in the ER, thereby
increasing the amount of Env glycoprotein available for transport to the cell surface.
Vpr
The vpr gene (for viral protein r) encodes a 14-kDa, 96 amino acid protein that is incorporated efficiently into virions. The incorporation of Vpr depends upon a specific interaction
with a Leu-rich motif located near the C-terminus of p6 (Fig. 7). In addition to weakly stimulating gene expression from the HIV LTR, Vpr also induces the arrest of Vpr-expressing cells in
the G2 phase of the cell cycle, and reportedly facilitates transport of the viral PIC to the nucleus
of infected cells.
Although it is not entirely clear why HIV would evolve a cell-cycle arrest function, a
number of groups have reported that Vpr rapidly and efficiently arrests cells in G2. Indeed, it
has been suggested that de novo Vpr expression is not required and that the Vpr present on
incoming virions is sufficient to induce cell-cycle arrest. G2 arrest appears to result from inhibition of the p34cdc2-cyclin B kinase complex.64 It has been proposed that HIV LTR expression
is increased during G2, thus perhaps providing a rationale for the evolution of this cell-cycle
arrest function.
28
Several observations have suggested that Vpr might play a role in nuclear import of the
viral PIC: 1) Vpr mutation reduces virus infectivity in fully differentiated monocyte-derived
macrophages, 2) Vpr is detected in PICs, and 3) Vpr, when expressed in cells, localizes to the
nucleus. A variety of models have been proposed to account for the ability of Vpr to stimulate
nuclear import; these will be discussed in more detail in the next chapter.
Vif
Expression of Vif (for viral infectivity factor) is highly conserved among lentiviruses; it is
encoded by all lentiviruses except equine infectious anemia virus. Vif mutation can cause profound defects in virus infectivity. Interestingly, the defective phenotype is cell-type dependent
and is determined not by the target cell but by the virus-producing cell. Thus, certain cell lines
(for example HeLa, COS, 293T, SupT1, CEM-SS and Jurkat) are permissive for Vif mutants; virus produced from these lines is fully infectious regardless of the target cell used. In
contrast, other cell types (most notably, primary lymphocytes and macrophages) are nonpermissive. This cell-type specificity argues that host factors play a role in Vif function, and
that the defect observed with vif defective mutants is imposed during virus assembly. Analysis
of transient heterokaryons formed between permissive and non-permissive cells has indicated
that the non-permissive phenotype is dominant, perhaps suggesting that Vif counteracts the
effect of a cellular factor that inhibits the formation of infectious virions.65
Although the Vif-defective phenotype may be imposed during assembly, it is manifested early
post-entry as a failure to efficiently reverse transcribe the viral genome. Reminiscent of observations made with certain post-entry Gag mutants, Vif(-) virions have been reported to display
defects in proper core condensation. Interestingly, Vif has been detected at low levels in virus
particles; however, the implications of virion incorporation for Vif function remain unclear.
Nef
Although Nef was originally reported to suppress gene expression from the HIV LTR (hence
the name negative factor) it is now clear that Nef plays an important positive role in lentiviral
pathogenesis. Nef is a 27 kDa, membrane-associated phosphoprotein; like Gag, its membrane
binding is dependent upon a myristic acid moiety covalently attached to the N terminus.
As is the case for the other HIV accessory proteins, several primary Nef functions have
been reported: 1) downregulation of CD4 and major histocompatibility class I (MHC I) molecules from the cell surface, 2) stimulation of virus infectivity in single-round assays, and 3)
modulation of cellular activation pathways. As mentioned above, Nef is one of the three viral
proteins (along with Env and Vpu) whose expression reduces cell-surface expression of CD4.
Nef-induced CD4 downregulation is achieved by increasing the rate at which CD4 is internalized from the plasma membrane66 reportedly via Nef acting as a bridge between CD4 and
adapter protein (AP) complexes in clathrin-coated pits. Nef also downregulates cell-surface
expression of MHC I molecules, perhaps impairing the ability of cytotoxic T lymphocytes
(CTLs) to detect and eliminate virus-expressing cells.67
Although in general the effects of Nef deletion on virus replication kinetics in culture are
quite limited, it has been reported that in single-cycle assays, the presence of Nef modestly
stimulates virus infectivity. Again, the Nef(-) defect is manifested by a reduction in the amount
of viral DNA synthesized post-infection. It is currently unclear to what extent this enhancement of virus infectivity contributes to the requirement for Nef expression in vivo.
It has been unambiguously demonstrated that Nef(-) clones of SIV display profound defects in virus replication and disease induction in infected macaques.68 The presence of nef
deletions in virus isolates obtained from at least some infected individuals who progress to
disease very slowly (the so-called long-term non-progressors) implies that Nef may play a similar role in maintaining high virus loads in HIV-1 infected humans. Although this requirement
HIV-1 Replication
29
for Nef in vivo remains unexplained, Nef contains a highly conserved consensus binding site
for Src homology region 3 (SH3) domains. Indeed, Nef has been reported to interact with a
variety of Src-like kinases and affect their activities.69 The effect of such interactions on signal
transduction pathways could stimulate virus replication in vivo.
Nef has been detected at low levels in virus particles, where it localizes to the virion core.
As is the case for Vif, the implications of these findings remain to be determined.
Vpx
Although the focus of this chapter is on HIV-1, it is worth noting briefly the functions of
an additional protein, Vpx, that is encoded by the genomes of the HIV-2/SIVsm/SIVmac lineage
of primate lentiviruses (but not by HIV-1). Vpx bears considerable sequence homology with
Vpr, and, like the latter protein, is incorporated at relatively high levels into virions via an
interaction with the C-terminus of Gag. Vpx appears to play a role in infection of non-dividing
cells but does not induce cell-cycle arrest.70 Thus, the proposed nuclear import/cell cycle arrest
functions of HIV-1 Vpr are segregated into two proteins (Vpr and Vpx) in the HIV-2/SIVsm/
SIVmac lentiviruses.
Concluding Remarks
During the past 15 years, a wealth of knowledge has been acquired concerning the replication cycle of HIV-1. However, it should be clear from the above discussions that much
remains to be learned before our understanding is complete. From the perspective of lentiviral
vector development, the mechanism by which HIV-1 infects non-dividing cells is of particular
interest. This topic will be the focus of Chapter 3.
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CHAPTER 3
Abstract
Introduction
Because retroviruses require integration into the host cell genome to complete the viral life
cycle, the virus must have a mechanism for getting its genome in proximity to the host cell
chromosomes. The oncogenic retroviruses require mitosis for integration, presumably because
this then allows them access to host cell chromosomes.1,2 However, one of the distinguishing
features of lentiviruses is the ability to infect non-dividing cells.3 Why lentiviruses share this
characteristic and how they are able to infect non-dividing cells is not well understood.
Macrophages, or cells of the monocyte lineage, are major in vivo targets of all lentiviruses.
Macrophages are terminally differentiated cells that are in a G0, or non-proliferating, state.
From a teleological perspective, lentiviruses either have the capacity to infect non-dividing
cells, and so macrophages became a major target; or because the lentiviruses need to infect
macrophages to complete their life cycles, they evolved mechanisms to infect non-dividing
cells. Interestingly, the primary targets for the primate lentiviruses (HIV and SIV) and FIV are
activated T-cells, a rapidly dividing cell type. Yet infection of non-dividing cells is important for
the life cycle and pathogenesis of these viruses. For HIV, non-dividing target cells include
macrophages which have been implicated in transmission of virus between hosts, and resting T
cells which have been implicated in transmission and in persistence of a viral reservoir.4 Whether
proliferating or not, cells must be metabolically active; completely quiescent cells cannot complete reverse transcription and are not productively infected by HIV.5 Thus even the lentiviruses
with expanded cell tropism depend on infection of non-dividing cells.
The proposed determinants for HIV infection of non-dividing cells, examining the evidence for each determinant and pathway and referring to relevance of HIV results for other
Lentiviral Vector Systems for Gene Transfer, edited by Gary L. Buchschacher, Jr.
2002 Eurekah.com.
34
lentiviruses will be presented. This list includes the viral structural proteinmatrix (MA), a
viral enzymatic proteinintegrase (IN), a viral accessory proteinVpr, a viral cis-acting DNA
sequencethe central polypurine track (cPPT), and the host cell cytoskeleton.
Nuclear Transport
Before discussing how viruses reach the nucleus, a brief overview of cellular nuclear transport will be given (Fig. 1). Bi-directional transport of molecules between the nucleus and cytoplasm of eukaryotic cells is controlled by large (approximately 125 MD), multi-protein complexes called nuclear pore complexes (NPC) that are found throughout the double layered
nuclear envelope which maintain the separation of these compartments.6 The aqueous channels permit the diffusion of small molecules into and out of the nucleus, but larger molecules,
usually anything greater than 50 KD or larger in diameter than 9 nm, require signal-mediated
transport.7,8 However, nuclear molecules smaller than the theoretical exclusion size usually
contain nuclear import signals. Nuclear import and export of proteins and RNAs are mediated
by a family of nuclear transport proteins, generally termed importins (or karyopherins) and
exportins (reviewed in ref. 8).9 In general, nuclear proteins contain a nuclear localization sequence (NLS) that is recognized, either directly by a member of the importin /transportin
family of transporters, or indirectly through an adapter protein which subsequently binds to
the importin for cargo transport. The most studied of these adapters is importin , which
mediates protein import via the classical pathway. Importin binds to a single or bipartite
stretch of basic amino acids that functions as a NLS. However, other adapter proteins have
been identified which recognize different NLSs. Nuclear export works in a similar way, but
with different transporter and adapter proteins.8,10 These pathways often involve the transport
of RNA or RNPs from the nucleus to the cytoplasm. As for nuclear import, different nuclear
export substrates use different nuclear export signals (NES) and transportins, but all pathways
converge at the nuclear pore. The best characterized nuclear exporter is CRM1, which mediates
nuclear export of leucine-rich NES cargo proteins by direct binding to the NES.11,12 Finally, there
are some nucleocytoplasmic shuttling proteins, best exemplified by the M9 signal of hnRNP
A1,13 containing overlapping import and export signals which often cannot be separated, and
so are termed nuclear shuttling (NS) signals.14
Directionality of nucleocytoplasmic transport is thought to be controlled by a gradient of
GTP and GDP bound forms of the GTPase, Ran.15-17 The Ran GTPase-activating protein
(RanGAP) and its activators are in high concentrations in the cytoplasm while the Ran guanine
nucleotide exchange factor (RanGEF) is at high concentrations in the nucleus, leading to the
prediction that cytoplasmic Ran is mostly GDP loaded and nuclear Ran is mostly GTP loaded.
For nuclear import, RanGTP promotes dissociation of importin and its cargo on the nuclear
side while nuclear export factors bind cargo at higher affinity in the presence of RanGTP
(nuclear) and release cargo in the presence of RanGDP (cytoplasmic). This model fits currently
available data to explain directionality of transport, but the mechanism for actual transit through
the pore is still not understood.18
35
Genome
Structure
Transport
(envelope/capsid size)
Nuclear Entry
to Nucleus
Adenovirus
dsDNA
non-enveloped
90 nm
capsid in endosome
& direct contact with
microtubule motors;
capsid disassembly
during transport
capsid binds
NPC, injects
DNA
through pore
SV40
dsDNA
non-enveloped
50 nm
capsid in endosome
& soluble import
factors
through NPC
after capsid
conformational
changes
enveloped 100 nm
through NPC;
unknown
mechanism
Influenza
minusstrand RNA,
segmented
enveloped 20X100 nm
long rods
RNA-RNP complexes
with soluble import
classical import
via NPC
factors
Oncogenic
Retovirus
RNA,
enveloped capsid 50X
with DNA
100 nm cones;
intermediate PIC 28 nm
PIC in cytoplasm,
unknown mechanism
at mitosis;
unknown
mechanism
Lentivirus
(HIV)
RNA,
enveloped capsid 50X
with DNA
100 nm cones;
intermediate PIC 28 nm
PIC in cytoplasm,
possible actin &
microtubule
cytoskeleton; soluble
import factors
through NPC;
mechanism
unknown
Many DNA viruses transport their genomes to the intact nucleus to take advantage of
host cell DNA replication factors. Some RNA viruses also transport their genomes to the nucleus,
not for RNA synthesis, but to make use of host cell splicing factors. The mechanisms for
transport are as varied as the viruses, but all pathways converge at the nuclear pore for entry.19
Transport of DNA is not a normal cellular function, and so viruses must adapt the normal
cellular nuclear import pathway to deliver their genomes. Viral genome complexes and capsids
are too large to diffuse through the cytoplasm,20 and so many of them enlist host cell cytoskeletal
transport systems, particularly microtubules,21 in addition to the soluble nuclear import factors.22 Two DNA tumor viruses, SV40 and adenovirus, use different mechanisms for delivering their genomes to the nucleus. However, both remain as intact capsids throughout much of
their cytoplasmic transit. The non-enveloped SV40 virion enters the cell by endocytosis and is
transported through the cytoplasm in the endosome. Near the nucleus, the viral particle, having adopted a modified conformation, exits the endosome. In this partially dissociated form
composed of viral structural proteins, the still recognizable viral particle enters the nucleus
through the NPC, facilitated by its NLS containing proteins.23 In contrast, adenovirus capsids
travel on microtubules, initially within an endocytic vesicle, but later, by direct interaction
with host cell microtubule motors.24,25 The adenovirus capsid docks at the NPC and then
36
Fig. 1. Nuclear import in the cell. The major nuclear import factors and pathways are represented schematically, beginning with NLS-substrates binding to soluble nuclear import factors (the adapter, importin and
the transporter importin or transportin) in the cytoplasm. These complexes are docked at the nuclear pore
and then translocated through the pore into the nucleus where they dissociate when RanGDP is converted
to RanGTP. Import factors are recycled to the cytoplasm via nuclear export pathways and NLS substrates
remain in the nucleus. The directionality is likely controlled by higher concentrations of RanGDP in the
cytoplasm and higher concentrations of RanGTP in the nucleus, maintained by the Ran GTPase activating
protein (RanGAP) and the Ran guanine nucleotide exchange factor (RanGEF), respectively.
dissociates as its genome is injected through the pore into the nucleoplasm.22,26 Interestingly,
the dissociation of the adenovirus genome from its capsid occurs in a stepwise fashion, beginning
immediately upon endocytic uptake of the virion as it enters the cell, and continuing until the
genome is completely dissociated from the capsid to enter the nucleus.24 Herpes virus capsid
enters the cytoplasm via direct fusion between the viral envelope and the cell plasma membrane. The released capsid is transported by dynein along microtubules to the NPC where the
capsid is left and the genome with associated viral proteins enters the nucleus.27 The RNA
genome of influenza virus enters the nucleus as part of ribonucleoprotein (RNP) complexes,
making use of the cellular nuclear import pathways by NLSs in the protein component of the
RNPs.28, 29 Thus delivery of a viral payload to the nucleus is not unique to retroviruses, but this
part of the life cycle has been uniquely difficult to study for retroviruses. The capsids of the
viruses described above remain largely intact, or at least as recognizable structures, throughout
much of this process. Retroviral particles disassemble, and the resulting complexes for reverse
transcription and integration are difficult to visualize and remain poorly defined throughout
the cytoplasmic transport and nuclear entry.
37
Fig.2. HIV and MLV preintegration complexes and nuclear entry. Structural (black circles) and enzymatic
proteins (white circles) are part of the viral core, and some remain associated with the PIC after viral entry,
uncoating and reverse transcription. The structure of the PIC is not well defined, but is known to differ in
composition between the lentivirus HIV (which has a partially triple stranded genome and carries the
accessory protein Vpr) and the oncogenic retrovirus, MLV. HIV infects in the absence of mitosis and
contains several potential determinants for nuclear entry, listed right, but MLV requires mitosis for integration and productive infection. Note, DNA flap drawn larger, out of scale, for emphasis. RT, reverse transcriptase; NC, nucleocapsid; CA, capsid; MA, matrix; IN, integrase.
38
39
Fig. 3. Lentivirus genomes. Note that the accessory genes, vpr/vpx are found only in primate lentiviruses and
that vif is found in all lentiviruses except EIAV (Modified from ref. 3).
40
41
Fig. 4. Involvement of cytoskeleton during early HIV infection. Many viruses (Table 1) depend upon host
cell cytoskeletal transport systems for delivery of the viral genome to the nucleus. This possibility has only
recently been investigated for HIV, and this diagram remains somewhat speculative.
these three proteins (MA, Vpr, IN) to MLV does not enable MLV to infect non-dividing cells
(A. Perez, unpublished data).72 The role of MA is in dispute. Vpr has nuclear import properties, but it is not part of non-primate lentiviruses. Although Vpr may have a facilitory function,
it is unlikely to be essential for HIV infection of non-dividing cells. Nuclear import functions
of Vpr and MA may be important for a different stage of the life cycle because both proteins
have been identified as nucleocytoplasmic shuttling proteins.63,73 IN has a demonstrable NLS,
the function of which appears important for HIV infection. However, it appears essential for
infection of dividing as well as non-dividing cells. How this might change our thinking about
determinants for infection of non-dividing cells is discussed further in the next sections.
42
Fig. 5. Models for lentivirus (HIV) nuclear transport and entry. These models are conjectural based upon
currently available data. A) Lentiviruses may require transport of the PIC through the cytoplasm to the
nucleus, making use of host cell microtubule transport. Microtubule motors would perform the actual
translocation, but it is unclear if motors would interact directly with viral components of the PIC or through
a cellular protein. B) IN is the best viral protein candidate for HIV nuclear entry. It may facilitate viral
genome entry through proposed classical NLSs or through more recently proposed functional nuclear
import signal that likely operates by binding to another protein with a NLS. This other protein, designated
X, would interact with the cellular nuclear import factors, such as importin . Protein X could be a viral
protein, such as MA, or more likely, an unidentified cellular protein. C) The DNA flap produced from the
cPPT is a structural element required for HIV infection, appearing to be important for nuclear entry. D)
All of the above proposed determinants may work together to promote nuclear entry of the PIC, enabling
infection of non-dividing cells. In this view, the complex may be transported to the nucleus on microtubules,
where soluble nuclear import factors and viral proteins, such as Vpr, dock it to the NPC. Nuclear import
signals in viral and cellular proteins associated with the complex and the genome structure promote
translocation of the complex through the pore, into the nucleus where it is able to integrate into the host
cell genome. Note, DNA flap drawn larger, out of scale, for emphasis. PIC, preintegration complex; IN,
integrase; , importin ; NPC, nuclear pore complex.
43
this structure for infection of non-dividing cells is that when transferred to a HIV vector, it
enhances transduction in non-dividing cells by about 10 fold.77,78 However there are problems
with interpreting this central DNA flap as the determinant for nuclear entry of the genome and
infection of non-dividing cells. Mutations in the cPPT affect infection of dividing as well as
non-dividing cells. Therefore the defect is not specific to non-dividing cells. The authors point
out that there is no direct evidence for a mitosis-dependent mechanism for lentiviral genome
nuclear entry and that a failure at nuclear entry may affect dividing as well as non-dividing
cells;74 this is similar to the phenotype of the newly described IN-NLS mutant above.71 Also, it
is unclear if this cDNA flap is a stable structure that is preserved up to integration.79 Additionally, the cPPT is not absolutely required for infection of non-dividing cells because HIV vectors that lack the cPPT still transduce non-dividing cells.80 Perhaps the most striking feature of
these studies is that they raise the possibility of a structural determinant for infection of
non-dividing cellsa structure found in lentiviruses, but not in oncogenic retroviruses.
44
even this is not absolutely required to infect non-dividing cells. It is striking that neither HIV1 IN or the cPPT transferred to MLV confers ability to infect non-dividing cells.
Most studies on lentiviral infection of non-dividing cells have focused on nuclear entry,
but perhaps nuclear entry per se is not the determining factor. This suggests that nuclear entry
may be necessary for infection of non-dividing cells, but some other determinant(s) may govern this process. Perhaps in analogy with infection of other viruses, the infection needs to be
viewed as a wholeviral entry, uncoating and reverse transcription, transport of the PIC,
nuclear entry of the genome, and integrationwith each step a necessary precursor to the
next. In this whole process, a hierarchy of necessary steps, lies the difference between lentiviruses
and oncogenic retroviruses, and while disruption of a single step will block lentiviral infection,
transfer of a single determinant will not enable oncogenic retroviral infection of non-dividing
cells. The gaps between entry at the plasma membrane and nuclear entry, and between nuclear
entry and integration may hold the keys to our understanding lentiviral infection of nondividing cells.
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CHAPTER 4
Abstract
uman immunodeficiency virus type 1 (HIV-1) based gene transfer systems are
gaining in popularity due to their ability to transduce terminally differentiated and
non-dividing cells. Oncoretroviral vectors based on Moloney murine leukemia virus
(MoMLV), on the other hand, can only transduce dividing cells. The reasons for increased
ability of lentivirus vectors to transduce such cells has been attributed to several of the viral
proteins (integrase, matrix and Vpr) that are purported to be involved in the nuclear import of
the pre-integration complex (PIC). Nuclear import is also augmented by a unique triple stranded
DNA region created during reverse transcription of the incoming viral RNA in the target cell
(discussed in chapter 3). This chapter deals with the rationale behind the design of human
immunodeficiency virus type 1 (HIV-1) based packaging systems with an emphasis on some
recent advances in the field for the creation of safe and efficient HIV-1 based vectors. The
review covers trans-acting proteins and cis-sequences required for the deployment of HIV-1
vectors for gene transfer. This is a rapidly advancing field that with further refinements may
soon allow the utilization of HIV-1 based and/or other lentivirus vectors in a clinical setting.
Introduction
Designing a gene transfer system using HIV-1 requires an understanding of key features
of the replicative cycle of the virus (see chapter 2 for details). A brief overview of HIV-1
replication is presented below emphasizing those aspects critical for the creation of an HIV-1
based packaging system. The overview is followed by a detailed description of the various
components of an HIV-1 based packaging systems including the various flavors it comes in.
49
Fig. 1. Schematic representation of the coding regions in HIV-1 provirus (A). The coding region of the viral
regulatory proteins, Tat and Rev, are each derived from two exons. The major 5' splice site is marked but
other splice donor and acceptor sites are not shown. Transcription from the 5' LTR of the provirus results
in single genome-length RNA (B) from which all other RNA species are derived. The full-length or
unspliced mRNA is used for encapsidation into the virus particle and is also the substrate for translation into
Pr55Gag and Pr160Gag-Pol precursor polyproteins (C). The viral proteins Vif, Vpr, Vpu and Env are translated
from singly spliced mRNAs while Rev, Tat and Nef are translated from multiply spliced RNAs. The various
subunits derived from the two precursor polyproteins are shown. Further details are provided in the text.
viral RNA to be produced in the infected cell. In the absence of Tat most transcripts suffer
premature termination.
The full-length message derived from the provirus is alternatively spliced to generate
multiple species of mRNA. At least 30 species of mRNA have been described in HIV-1 with
several slightly different mRNAs coding for the same protein.6-13 The fully spliced mRNAs
encode for Rev, Tat or Nef. The unspliced message codes for Gag and Gag-Pol proteins while
the singly and partially spliced messages code for envelope (Env) and the accessory proteins
Vif, Vpr or Vpu. Since incompletely spliced messages are usually retained in the nucleus of
eukaryotic cells, the expression of proteins derived from these messages requires that the corresponding RNAs exit the nucleus without undergoing splicing.14-17 Moreover, sequences have
been identified in gag that retain the unspliced message in the nucleus.18-20 The inhibitory
sequences have been referred to as cis-acting repressor sequence (CRS) elements or cis-acting
inhibitory sequences (INS). The Rev protein binds to a target sequence within the env coding
region called Rev-response element (RRE) to bring about nucleo-cytoplasmic transport of such
messages. Rev may also play a role in the stability of the RNA and its polysomal association or
translation.21,22 Studies have indicated that for Rev to bring about nucleo-cytoplasmic transport, the RNA must form a complex with the splicing machinery. Thus, an intact 5 splice
50
donor site in the RNA is essential for Rev to produce its effect.23 The 5 splice donor is also used for
the generation of partially spliced messages that are translated into Vif, Vpr and Vpu proteins.
Virion Assembly
HIV-1 is an enveloped virus. The surface of the virus particle is decorated with the envelope (Env) glycoprotein consisting of a surface glycoprotein (gp120) and a transmembrane
protein (gp41). Lining the inner aspect of the lipid envelope is the viral matrix (MA/p17)
protein. The most characteristic structure of the virus is the cone shaped core composed predominantly of the capsid (p24) protein. Also present within the virion are the nucleocapsid
(NC/p7) protein , and the viral enzymes consisting of the reverse transcriptase (RT), integrase
(IN) and protease (PR). The internal proteins of the virion are derived for the most part from
the Gag (Pr55gag) and GagPol (Pr160 gagpol) precursor polyproteins (Fig. 1). Both precursors are
translated from the unspliced mRNA molecule containing the gag and pol coding sequences.
The Pr160Gag-Pol is synthesized by a ribosomal frame-shifting mechanism that occurs during
translation of Gag once every 10 or 20 translation events.1,24 The Gag precursor can assemble
into virus-like particles in the absence of other internal proteins of the virion.25-35 The Gag-Pol
precursor, while it cannot form virus particles on its own, is drawn into the assembling particle
through its interaction with the Gag precursor.36-39 The enzymes present as part of the Gag-Pol
precursor, PR, RT and IN, are thus incorporated into the virus particle.
Vpr and Vif have also been demonstrated within the virion. Vpr is incorporated into
assembling virus particle through an interaction with the p6 protein present in the C-terminus
of the Gag polyprotein.40-42 Vif is incorporated into the virion via an interaction with the
genomic RNA.43
The precursor polyproteins, together with two molecules of genomic RNA, accumulate
beneath the portion of the plasma membrane containing the viral Env glycoprotein. A viruslike particle then pinches off from the plasma membrane. The genomic RNA is incorporated
into the assembling virion via interactions between the nucleocapsid (NC) protein within Gag/
Gag-Pol proteins and the encapsidation or packaging (E/) signal present in the vector RNA
molecule. Following release of the virus particle, the Pr160Gag-Pol precursor is cleaved to yield
the viral PR, RT and IN proteins. Proteolytic cleavage of the Gag precursor yields the MA
(p17), CA (p24), NC (p7) and p6 proteins. The mature virus particle is then ready to infect
target cells.
Virus Entry
Following binding of the virus to the target cells via the Env glycoprotein and corresponding receptor/coreceptors on the target cell, fusion of the viral and plasma membrane of the cell
ensues. This results in the deposition of the viral core into the cytoplasm. The core is stripped
of the capsid protein and the genomic RNA is reverse transcribed into a double stranded cDNA.
During reverse transcription, due to presence of a central polypurine tract (cPPT) and termination sequence (CTS), a unique triple-stranded DNA region consisting of the double stranded
DNA and a DNA flap is created in the middle of the newly synthesized cDNA. This unique
structure, by an unknown mechanism, enhances the import of the viral pre-integration complex (PIC) into nucleus of the target cell.44-46 The PIC also contains the viral IN protein, MA
and Vpr proteins. These proteins may also be involved in nuclear import of the PIC although
the role of the MA protein in nuclear import is controversial. The cDNA is then inserted into
the chromosome of the target cell, apparently at a random location, by the viral IN protein.
51
Fig. 2. A schematic representation of the components of an HIV-1 based vector system consisting of a
packaging or helper construct (A), an Env expression construct (B) and a gene transfer vector (C). The
various RNA species derived from each expression construct are shown. Rev is required for expression of
helper and gene transfer vector RNA, both of which contain the RRE. Tat is required for expression of the
gene transfer vector RNA from the viral LTR promoter. The various components (proteins and two copies
of the vector RNA) assemble into a virus particle at the plasma membrane. Once budded off from the
membrane, the virus is then ready to infect and transduce the gene transfer vector together with its encoded
transgene into target cells.
gene transfer system using HIV-1 poses several unique challenges. This is because of the complexity of gene expression in HIV-1 (see above and chapter 2). For instance, expression of both
helper and gene transfer vector RNAs require sequences that ensure the transport of the RNA
molecules into the cytoplasm. To create a packaging system with HIV-1, one needs a) packaging or helper constructs that provide all necessary virion proteins to form a virus-like particle
and b) a gene transfer vector that provides the RNA for encapsidation by the assembling virus
particle (Fig. 2). The gene transfer vector ideally should expresses only the transgene of interest
but none of the viral proteins. The helper proteins consist of HIV-1 proteins that form the
internal proteins of the virion and an Env protein, either from HIV-1 or, more commonly,
from a different virus, which allows the virus particle to infect target cells bearing the appropriate receptor. The basic components of an HIV-1 based packaging system are described below.
52
Fig. 3. Cis-elements required for creation of an HIV-1 based gene transfer vector. The putative secondary
structure of the E/ signal that encompasses the entire 5' untranslated region is depicted. Elements involved
in reverse transcription and integration: primer binding site (PBS), polypurine tract (PPT),central polypurine
tract and central termination sequences (cPPT/CTS) and integration (att sites) are shown. The cPPT/CTS,
while not absolutely essential for the reverse transcription step in the context of lentivirus vectors, improves
nuclear translocaton of the pre-integration complex (PIC) in both dividing and non-dividing cells. The left
att site is derived from the u3 region present in the RNA genome while the right att site is derived from the
u5 region in the RNA. The RRE is required for nucleo-cytoplasmic of the vector RNA. Several different
lengths of RRE can be used. The nucleotide positions shown correspond to the extended RRE described
by Mann et al.208 : packaging signal. The transgene expression cassette is usually positioned between the
3' Tat/Rev splice acceptor site and the 3' LTR. Details of the cis-elements are discussed further in the text.
The sequences and numbers shown are with reference to the molecular clone pNL4-3 (GenBank Accession
number M19921).
53
Transcription Signals
Transcription of the vector RNA requires the enhancer and promoter elements in the U3
region and the R region in the 5LTR. The R region contains the TAR element that mediates
the transactivation by viral Tat protein to generate abundant amounts of genome-length RNA.
Encapsidation Signals
In order for the vector RNA to be encapsidated into the assembling virus particle, the
vector must contain an E/ signal. The entire 5' untranslated region constitutes the E/ region
and exhibits a complex secondary structure (Fig. 3). This region consists of seven stem-loop
structures. Four of these stem-loop (SL) structures encompassing the 5 splice donor site and
the 5' end of gag have been termed SL1, SL2, SL3 and SL4.49 SL1, SL3 and SL4 are involved
in Gag binding while SL2 contains the 5' splice site and is not essential for encapsidation. SL1
also contains the dimer-linkage signal (DLS) in its loop. Due to its location, SL1/DLS is present
in all HIV-1 RNAs, i.e., it is present in both spliced and unspliced RNAs. Deletion or mutations
in SL1 or SL3 reduces encapsidation of the RNA. Combined deletion of SL1 and SL3 has a
greater effect then deletion of each SL by itself. Studies have revealed that sequences upstream
of the 5 splice donor site including the lower stem of TAR and the poly A stem-loop are also
essential for packaging.50-53 It should be emphasized that mutations to the E/ region or
structures do not completely eliminate packaging but instead allow spliced RNAs to be packaged
in direct relation to their intracellular concentration.51,54,55 This is of concern because most
packaging systems designed to date contain a deletion within the so-called E/ region present
in the packaging or helper plasmid with the hope that the RNA derived from the helper construct
will not be copackaged with the vector RNA. The good news is that in the presence of RNA
with an intact E/ region, this RNA is likely to be preferred over the RNA with deletions in the
E/ region. However, it also appears that sequences outside of the E/ region can influence
encapsidation and or dimerization. For instance, presence of foreign sequences can negatively
affect encapsidation of vector RNA. Foreign sequences have also been shown to enhance
dimerization of RNA. Although the encapsidation and dimerization sequences overlap, they
can be functionally delinked from each other. Thus mutations to the dimer linkage site can
affect infectivity without affecting encapsidation.54 Thus more careful analysis of encapsidation
and dimerization of RNA present within vector particles is required to evaluate the effect of the
deletions or mutations within the E/ region of the helper construct on its encapsidation and
delivery to target cells.
gag Sequences
Studies in MoMLV suggested that the encapsidation signal extends into the 5' portion of
the gag.56 This appears to be true in the case of HIV-1 also. The SL4 structure of the HIV-1
encapsidation signal is present in the extreme 5' end of the gag coding region.49 While at least
40 nucleotides of gag are required for optimal packaging of the vector RNA, inclusion of
54
Polyadenylation Signal
The polyadenylation (pA) signal for the vector RNA is derived from the 3' LTR and the
core AATAAA sequence in HIV-1 is present within the R region. Several enhancers of pA have
been identified in the upstream U3 region using artificial constructs.67-69 It appears that most
of the U3 sequence can be deleted without compromising titer which in turn suggests that the
effect of these pA enhancers are not profound in the context of HIV-1 vectors.70,71 This aspect
is discussed in greater detail under self-inactivating (SIN) vectors
55
Tat
Tat is not required for expression of viral helper proteins but is required for the expression
of the gene transfer vector RNA (see above). Tat is, therefore, coexpressed along with the other
helper plasmids for production of virus stocks. The Tat protein is encoded in two exons. However recent experiments have shown that only the first coding exon of Tat is sufficient for
production of vector stocks for gene transfer.63,88 Tat has also been implicated in causation of
56
Nef
The Nef protein is synthesized early after infection together with Tat and Rev and has
pleiotropic effects. Nef has been shown to down modulate cell-surface expression of CD4 by
inducing endocytosis, resulting in the degradation of this molecule in lysosomes.127-130
Coexpression of Nef during virus stock production increases efficiency of gene transfer into
target cells.131-134 This has been attributed in part to its positive effect on increasing the efficiency of reverse transcription during viral entry.134,135 Fortunately, this effect of Nef is restricted to certain types of Env proteins used for pseudotyping virus. For instance, Nef increases infectivity of virions pseudotyped with HIV-1 or amphotropic MoMLV Env proteins
but not those pseudotyped with VSV-G or Ebola virus Env proteinss.132,136,137 Thus Nef is not
required for vectors pseudotyped with Env proteins that effect low pH mediated fusion and
entry of viruses. Nef can, therefore, be eliminated from packaging systems that employ VSV-G
57
Fig. 4. Schematic representation of simple gene transfer vector systems based on HIV-1. In A, a transgene
expression cassette consisting of a heterologous promoter driving a marker or therapeutic gene is inserted
into the env coding region and thus inactivates it. To produce infectious virus one needs to complement the
above construct with a separate expression construct for Env (B). The transduced target cell not only
expresses the transgene but also all other viral proteins with the exception of Env. A replication competent
gene transfer vector is shown in C. The transgene in this vector is positioned within Nef and is expressed
from a spliced mRNA. The expression of the transgene is, therefore, under control of the viral LTR promoter.
The vector produces all viral proteins with the exception of Nef.
58
Fig. 5. Schematic representation of an HIV-1 vector system 59 consisting of a first generation packaging
plasmid (A), gene transfer vector (B) and an Env expression construct (C). The packaging plasmid encodes
for all HIV-1 proteins with the exception of the Env protein. The 5' LTR is replaced with a heterologous
promoter and the 3LTR is replaced with a heterologous poly A (pA) signal. A deletion is engineered into
the region (). The gene transfer vector does not express any of the viral proteins and contains all cissequences for packaging, reverse transcription and integration (Fig. 2). It contains a transgene expression
cassette containing a therapeutic or marker gene under control of a heterologous internal promoter. An Env
expression construct (C) is provided for pseudotyping the vector to allow infection of target cells containing
the appropriate receptor to which the Env protein can bind.
59
Fig. 6. Schematic representation of a second generation HIV-1 packaging system,72,106,143,144 along with a
vector and Env-expressing construct. This is similar to the first generation packaging system (Fig. 5) but for
the elimination of the viral accessory proteins Vif, Vpr , Vpu and Nef.
60
Fig. 7. Schematic representation of a third generation HIV-1 packaging system.145 This is a minimal HIV1 packaging system that consists of three helper plasmids: a Gag/Gag-Pol expression construct (A), a Rev
expression construct (D) and Env construct (C) and a gene transfer vector. The expression of Gag/Gag-Pol
and the gene transfer vector RNAs requires the coexpression of Rev. Rev is produced using a separate
expression construct (D). The requirement for Tat for production of large amounts of gene-transfer vector
RNA is obviated by using a Tat-independent vector (B) in which the 5LTR is replaced with a chimeric
promoter consisting of heterologus promoter/enhancer elements substituting the corresponding viral elements (see also Fig. 10).
Rous sarcoma virus or from cytomegalovirus immediate early promoter (see below). The use of
four plasmids instead of the three and the elimination of Tat increases the safety of the system
by further decreasing the probability of recombination between the various helper plasmids
and the gene transfer vector to recreate a replication competent virus.
61
Fig. 8. Design of packaging systems based on the type of RNA-transport elements used in packaging and
gene transfer vectors.88 In the RRE/Rev-based packaging system (A) expression of RNA from both helper
plasmid and gene transfer vector is regulated by Rev and RRE. Rev coexpression is required for expression
of the helper and vector RNAs. In the CTE-based system (B), helper and vector RNA expression is regulated
by the MPMV-CTE. This is a Rev-independent packaging system i.e., no Rev coexpression is required for
production of vector stocks. In the combination packaging systems (C), the helper plasmid is regulated by
CTE while the gene transfer vector is controlled by RRE and Rev. An alternative scenario in which the helper
plasmid is regulated by RRE and Rev while the gene transfer vector is controlled by CTE is not shown. Rev
coexpression is required for production of vector stocks in this system. Possible sites of recombination
between helper and vector constructs in the three types of packaging systems are shown using bi-directional
arrows. Note that in C, the two constructs share homology only in the gag region. This design may be safer
than other packaging systems for production of vector stocks. For clarity, constructs expressing Env, accessory and regulatory proteins are not depicted. Adapted from: Srinivasakumar N, Schuening FG. J Virology
1999; 73: 9589-9598.
of CTE gives reasonable titers in some studies63,88,133,150,151 while other studies have reported
lower titers.106,149 The reason for these differences between the different studies is not yet clear.
There are two regions of homology between the helper and gene transfer vector constructs. One is at the 5 end of gag and the other is the shared RRE or CTE towards the 3 end
of the helper construct and the same element present within the gene transfer vector. Depending
on the RNA transport element being used, one can design three types of packaging systems for
production of HIV-1 vector stocks88 (Fig. 8) . The traditional or classical HIV-1 packaging
system uses the RRE and Rev for the expression of both helper and gene transfer vector RNAs.
The second packaging system uses the MPMV-CTE for expression of helper and gene transfer
vector RNAs. A possible utility of a CTE-based Rev-independent HIV-1 packaging system is
for the delivery of dominant negative forms of Rev into HIV-1 susceptible cells.133,150,151 In a
62
Fig. 9. Trans-lentiviral packaging system.155 In this packaging system, the proteins encoded in the gag and
pol coding regions are segregated in two different expression plasmids. One plasmid encodes for the Gag and
Gag-Protease (A) while a separate construct encodes for a Vpr-RT-IN fusion protein (B). The Vpr-RT-IN
is expressed using the LTR and RRE from HIV-2. This is based on the observation that Tat and Rev proteins
of HIV-1, encoded in the Gag-Pro expression construct, can also function in the context of HIV-2 LTR and
RRE-2. The gene transfer vector (C) and an Env expression construct (D) are the other components of the
packaging system required for the production of vector stocks.
Rev-RRE based system, the coexpression of a dominant negative Rev as part of the gene transfer
vector would be inhibitory for vector stock production. The third kind of packaging system is
called the combination or reciprocal packaging system and utilizes the Rev and RRE for
expression of one component of the packaging system and the CTE for the other
component.88,151 Using dissimilar transport elements for the expression of the helper plasmid
and the gene transfer vector RNAs in the combination packaging system may render the
packaging system safer by reducing the chance of replication competent retrovirus (RCR) formation.
Packaging System Using Codon-Optimized Helper Construct
As an alternative approach to decreasing the homology at the gag end of the helper and
gene transfer vector constructs, some investigators have constructed a helper plasmid containing a humanized gag coding sequence.153 Interestingly, this not only reduces the homology in
the gag region due to the silent mutations introduced, it also renders gag expression Revindependent. Elimination of homology at the gag end and at the 3 end by removal of RRE in
the helper plasmid in most probability makes this packaging system one of the safer systems
63
described to date. On the other hand, a non-homologous recombination between the helper
and gene transfer vector involving the gag region can result in a recombinant that may be able
to express the Gag protein in target cells even in the absence of Rev. One can envisage the use
of such codon optimized packaging system for the delivery of antisense RNA expression cassettes targeted to the gag and/or env coding regions of HIV-1. It may be possible to use this
system to deliver dominant negative Rev into target cells providing that the inhibitory effect of
dominant negative Rev on vector RNA transport in the producer cell can be overcome using the
CTE or other modifications to vector backbone.
Vector System Using Combination of Two Different Lentiviruses
Another approach for decreasing homology between the components of a lentivirus packaging system is to use a helper construct derived from SIV to package an HIV-1 gene transfer
vector.154 Currently, such packaging systems are still in their infancy and need to be developed
further. For instance, the titer of this system is about an order of magnitude less than that
obtainable with an Rev-RRE based HIV-1 packaging system. However such novel systems
have great potential because of other benefits. For example, the Vpr of HIV-1 can increase
efficiency of infection of macrophages. But Vpr of HIV-1 has the disadvantage of producing
cell-cycle arrest and/or apoptosis of target cells when large amounts of protein are delivered via
virus particles. In SIV, these functions of HIV-1 Vpr are segregated between two different
proteins, the SIV Vpr and Vpx.1 The SIV Vpx, like HIV-1 Vpr, can augment nuclear import of
PICS but without the apoptotic or cell-cycle arrest phenotype. This latter function is relegated
to the SIV Vpr protein. Thus one can create an SIV based helper plasmid that encodes for Gag,
Gag-Pol and Vpx but not Vpr and use this for generating HIV-1 vector stocks. The alternative
system containing HIV-1 packaging construct and SIV based gene transfer vector has also been
described.62 The latter packaging system does not have some of the advantages of the former,
namely the use of SIV Vpx instead of HIV-1 Vpr.
Packaging System Using Separate Helper Plasmids for Expression of Gag and Pol
Coding Regions
A recent novel modification to the packaging system involves a clever approach to separate gag and pol coding regions.155 In this novel packaging system (Fig. 9), gag/protease is expressed
using one construct. The other enzymes of the Gag-Pol precursor, namely RT and IN are
expressed as a Vpr-RT-IN fusion protein using a separate plasmid construct. The Vpr-RT-IN
fusion protein is drawn into the assembling virus particle through its interaction with the p6
domain of the Gag precursor (see Virus Assembly above). Thus the internal proteins of virus
particle are derived from two separate plasmid expression constructs. The use of separate plasmids for gag and pol proteins provide a higher margin of safety and has been shown to predictably
reduce the frequency of recombination between the packaging plasmids and gene transfer vector.
64
Fig. 10. Modifications to HIV-1 gene transfer vector to improve safety and efficacy. Details are provided in
the text. Adapted from Srinivasakumar N. Packaging cell system for lentivirus vectors. In: Morgan J, ed.
Gene Therapy Protocols, 2nd ed. 2001, Humana Press.
vectors. Sin vectors were first described for MoMLV and SNV vectors.156,157 These studies
revealed that mutations in U3 frequently resulted in lower vector titers; this observation was
explained by the requirement of some of the sequences in this region for efficient polyadenylation
of vector RNA. In HIV-1, the core pA signal (AAUAAA) is present within the R region.
Sequences in the U3 region have been identified in HIV-1 that also modulate the efficiency of
poly adenylation.67,69,158 The most important of these sequences is present between the TATA
box and the R region and is about 20 bp in length. In contrast to what has been observed with
MoMLV vectors, it appears that most of the U3 region of the 3 LTR ,with the exception of the
poly A enhancer described above and the attachment (att) sequence at the 5end of U3 recognized by viral IN, can be safely deleted without compromising vector titer.70,71 Such a deletion
ensures that transcription from the 5 LTR promoter is efficiently suppressed following reverse
transcription and integration into the target cell chromosome. Another advantage of using
vectors with deletions in the U3 region is the enhanced transgene expression noticed from
some internal promoters in such vectors.65,71,159 This is probably due to a decrease in promoter
competition between the viral LTR and the internal promoter.160
Tat-Independent HIV-1 Vectors
Tat is not required for expression of helper proteins but is essential for obtaining high
levels of transcription from the viral LTR promoter of the gene transfer vector. Several studies
have shown that titers are lower if Tat is not provided during virus stock production.133,145,149
To overcome this, hybrid promoters that use enhancer elements from other viruses such as
cytomegalovirus immediate early promoter and Rous sarcoma virus LTR, instead of those present
in the U3 of HIV-1, have been created.145, 149 These vectors appear to be nearly as efficient in
terms of vector titer as the original Tat-dependent vectors that contained the wild type HIV-1
LTR. Although there are reports showing that Tat, in addition to its effect on transcription
from the viral LTR, can also effect the efficiency of reverse transcription,161 studies using HIV1 vectors have not revealed this requirement.145,149 Despite the observation that the requirement for Tat can be overcome by using hybrid promoters, it may not be possible to entirely
65
remove the TAR element due to the presence of sequences essential to ensure optimal packaging
of vector RNA.53,162 Moreover, the R region, including TAR, is essential for reverse-transcription.
Rev-Independent HIV-1 Vectors
The CTE from MPMV can substitute for Rev and RRE function in proviral clones, packaging plasmids and in gene transfer vectors (see above). To create a Rev-independent gene
transfer vector, this small RNA element of about ~200 bp is usually inserted between the
transgene expression cassette and the 3' LTR63,88,133,150,151,163-165 (Fig. 10). If the CTE is placed
further upstream, say in place of the RRE, its function seems to be compromised. Some investigators have hypothesized that the CTE needs to be as close as possible to the pA signal (< 200
nucleotides) to exert its effect.165 A Rev-independent vector is ideal for transducing
transdominant Rev-encoding transgenes into HIV-1 susceptible cells.133,150 The drawback of
this vector is that recombination with the helper construct involving the gag/pol region could
allow Rev-RRE independent expression of Gag-Pol proteins in the transduced target cells.
Minimal Gene Transfer Vectors
The two important reasons for attempting to create a minimal gene transfer vector are: 1)
to decrease regions of homology between the gene transfer vector and packaging plasmid and
2) to increase the payload carrying capacity of the vector. It is believed that it may be possible
to accommodate between 8 and 11 kb size of foreign sequence within an HIV-1 vector.47,48
Therefore, the smaller the size of the vector backbone, the larger the size of the insert that it can
accommodate. One of the smallest vectors described to date 61 has about 550 bp of the 9.7 kb
of the HIV-1 provirus genome. This vector contains the 5LTR and the entire 5' untranslated
region upstream of gag and about 40 nucleotides (nt) of gag. The 5' major splice donor site in
the 5' untranslated region is mutated. The vector lacks the RRE and contains deletions in U3
and U5 regions of the 3LTR. The pA functions in the U5 region is restored using the bovine
growth hormone pA signal. This vector has approximately 50% of the titer of the wild-type
vector. Insertion of the cPPT and CTS sequence in this vector (~170 nt) will probably improve
the titer obtained with this minimal vector without significantly affecting the payload capacity.
Most vectors used to date, in contrast, contain the 5 splice donor, an extended gag (350 to 500
nt), the cPPT and the RRE since the addition of these features usually provides higher vector
titers.
Vectors with Woodchuck Post-Transcriptional Regulatory Element (WPRE)
for Enhanced Transgene Expression
An element with Rev and RRE like properties is the woodchuck post-transcriptional regulatory element (WPRE).166 While the WPRE can substitute for Rev and RRE function in a
reporter construct that is widely used for assessing transport of intron containing messages, it is
not known if this sequence can substitute for Rev and RRE in the context of gene transfer
vectors or HIV-1 packaging constructs. Interestingly, experiments have revealed that addition
of the 600 bp WPRE in retroviral and lentiviral vectors downstream of the transgene can
increase expression by 5-8 fold 65,167(Fig. 10). This element may be of use to improve expression from weak promoters that may not function at optimal levels in some target cells.
66
interfering (DI) virus. HIV-1 vectors can interfere with the replication of wild-type virus by
several mechanisms. One mechanism is by competing and sequestering viral regulatory
proteins170 and the other is by competing with wild-type RNA for encapsidation into the
assembling virus particle.171 The presence of the vector in cells was found to interfere with the
spread of the wild-type virus in culture. During the spread of the wild-type virus, the vector
sequences are also likely to be mobilized to other cells where they can be expected to continue
to exert their interfering phenotype.
HIV-1 vectors can also be harnessed to deliver dominant negative proteins or RNA into
HIV-1 susceptible cells. The trick to using HIV-1 vectors for delivering interfering proteins or
antisense RNAs is to design packaging systems that will not be affected by expression of these
anti-HIV therapeutics in the vector producing cells. Even with packaging systems currently in
use, it should be possible to deliver antisense RNAs directed against the HIV-1 env region since
most packaging systems use Env from a heterologous virus for production of vector stocks.
Likewise one may be able to use gag antisense molecules in packaging systems using codonoptimized helper constructs as long as the antisense sequence does not overlap the gag portion
still present in the gene transfer vector. Finally, one can use a Rev-independent packaging
system where nucleo-cytoplasmic transport and expression of both helper and gene transfer
vector RNAs is regulated by MPMV-CTE to deliver interfering Rev into HIV-1 susceptible
cells.63,88,133,150,151 It is debatable if the use of HIV-1 based vectors for delivery of some of these
therapeutic genes has any advantages over using an unrelated lentivirus (discussed elsewhere in
this book) to achieve some of the same goals.
A novel approach to exploit interactions between the wild-type virus and vector sequence
has been suggested.58 This approach consists of expressing dominant negative proteins or antigenic epitopes of common infectious agents such as the hemagglutinin epitope in the HIV-1
vectors under control of the viral LTR promoter. When a wild-type virus infects the cell, Tat
produced from the incoming virus, transactivates the vector leading to the synthesis of dominant-negative protein or antigen. Cytotoxic T- cells directed against expressed antigen on HIV1 infected cells, if present, would then be expected to clear the antigen bearing cells. Alternatively, the dominant negative protein would be expected to interfere with replication of HIV-1
in that cell.
67
express physiological amounts of the transgene and/or express the transgene in only the tissue
of interest i.e., expression that is regulated in a tissue/cell specific manner. One excellent example
of this is a recent report using a lentivirus vector for erythroid-specific expression of human globin.168 In this vector the -globin gene was expressed using the -globin promoter together
with the upstream regulatory regions consisting of DNAse I hypersensitive sites that are known
to confer erythroid-specificity of gene expression. The entire transgene expression cassette was
inserted in an inverse orientation to that of the vector. Such an orientation allowed the retention of splicing signals within the expression cassette leading to significantly higher levels of
transgene expression than that observed with other vectors. Because the transgene expression
was restricted to erythroid cells, there was no negative impact on vector titer due to an antisense
effect in the producer cell. This vector provided therapeutic levels of -globin expression in
thallasemic mice. Reports such as this auger well for the development of lentivirus vectors
containing cell-type specific promoters for expression of transgenes.
Other modifications to HIV-1 vectors will be in the use of regulatable promoters that can
be turned on or off using small molecules such as tetracycline,182 Rapamycin, estradiol or
RU486.183-191 The drawback of the latter approach is that one will have to coexpress the protein that is required for gene regulation (e.g., tetracycline regulated transactivator) along with
the transgene. It is not clear how these regulators will affect functioning of the cell or whether
expression of these proteins will lead to immune-mediated elimination of the transduced cells.
Such vectors will eventually allow precise control of transgene expression in vivo. Precise control of transgene expression may be of particular importance in the nervous system where overexpression of neurotransmitters could lead to neurological effects.169
Site-Specific Integration
Due to the random nature of integration, lentiviruses have the some of the same disadvantages as other retroviruses, such as the possibility of either inactivating cellular genes (e.g.,
tumor suppressor genes) or activating or overexpression of other genes (e.g., oncogenes). Inadvertent activation of cellular genes, including oncogenes, can be avoided by using later generation of self-inactivating vectors which are devoid of promoter and enhancer elements in the
viral 3 LTR (see above). Alternatively, it may be possible to overcome the drawback of random
integration by redirecting PICs to specific regions of the host chromosome by fusing sequence
specific DNA-binding domains to the viral integrase.192,193
68
Safety Issues
Safety issues for the deployment of HIV-1 vectors for gene therapy in humans are discussed further in Chapter 8. Some aspects of vector safety are elaborated upon here.
Recombination between helper and gene transfer vector molecules can occur at the level
of input DNAs in the producer cells or can occur during reverse transcription as consequence
of copackaging of vector and helper RNAs or vector and cellular RNAs in the same virus
particle. One favored site of recombination between RNA molecules during reverse-transcription appears to be in the poly A tract of the helper RNA and the sequences upstream of the 3'
U3 and PPT in the gene transfer vector.155 The upstream recombination appears to occur, as
anticipated, in the gag region, which results in the joining of the 5LTR and packaging sequence of the gene transfer vector with the gag ORF of the helper plasmid. Whatever the
mechanism of recombination, the consequence is the transduction of molecules into the target
cells that may have an intact open reading frames for one or more viral proteins such as GagPol and/or Tat. Can these cells, harboring gag/pol sequences, produce viral proteins? Will this
lead to an immune response in the transplant recipient against the proteins expressed by the
recombinant vector? In other words, will the person be rendered HIV positive as deduced from
antibody assays? Another concern is that the reverse-transcription of the vector RNA is error
prone. It is likely that some of the vector genomes introduced into target cells will contain
deleterious mutations. These mutations (e.g., deletions, insertions or duplications) can lead to
inactivation of the transgene or synthesis of aberrant or nonfunctional proteins. What is the
effect of introducing these altered DNA sequences into target cells? Finally, vector genomes can
interact with wild-type HIV and possibly with endogenous retroviral sequences or elements.
What are the consequences of the interactions between vector and wild-type HIV-1 or endogenous retroviruses? Will this lead to the mobilization of vector or host-sequences from one cell
to another? Can novel viruses emerge as a result of recombination between vector and wildtype sequences or endogenous retroviral sequences? The answers to many of these questions
can only be ascertained by vigorous and careful evaluation of the vectors in large animals.
Preliminary studies from some investigators appear to indicate that HIV-1 based vectors are
safe in primates.204 But more careful studies are clearly needed.
How does one evaluate the safety of lentivirus vector stocks? The Center for Biologics
Evaluation and Research (CBER) of the Food and Drug Administration (FDA) recommends
that 5% of each vector supernatant lot and 108 or 1% of the end of production cells (which
ever is less) be tested for RCR for MoMLV vectors.205 There is, at present, no agreement on
what standards to apply for lentivirus vectors, particularly those based on HIV-1. It would
need to equal, if not exceed, those standards designed to detect RCRs in MoMLV vector stocks.
Also, there are no agreed upon assays to detect RCRs in HIV-1 vector stocks. The methods
commonly used are:
1. infection of target cells (including phytohemagglutinin-stimulated human peripheral blood
mononuclear cells) with vector stocks and assaying supernatant periodically for HIV-1 p24
over a two to four week culture period.204,206
2. Infection of indicator cell lines harboring lacz gene (Magi-cell assay)207 or puromycin resistance gene under control of HIV-1 LTR.155
69
3. Marker mobilization assays.155 The first method (assay for p24) can only detect replication
competent HIV while the latter two (infection of indicator cell lines and marker mobilization) can detect partial recombinants. The Magi-cell assay requires the recombinant to synthesize the HIV-1 Tat protein and thereby turn on lacz expression from the HIV-1 LTR.
Marker mobilization detects recombinants that can synthesize Gag-Pol , Tat, Rev and Env.
The last method can be modified to detect partial recombinants that can synthesize Gag-Pol
after appropriate modifications to the assay.155 Once the safety concerns are addressed, then
the first steps towards deploying HIV-1 vectors for clinical use can be taken.
Conclusions
There has been tremendous progress in the development of HIV-1 based vectors in the
last five years in terms of their safety and efficacy to deliver genes to a wide variety of tissues
both in vitro and in vivo. It is a vector system that shows great promise. However, the stigma
associated with HIV-1 based vectors, the lack of standardized assays for detection of recombinant replication competent HIV-1 emerging during virus stock production and the risk of
immune-mediated rejection of cells bearing a foreign transgene by the host are significant
hurdles that need to be addressed before clinical trials using HIV-1 vectors can be undertaken.
Acknowledgements
I wish to offer my apologies to those authors whose work was not cited due to an oversight
or because of space considerations. I wish to thank Gary Buchschacher, Jr. for his immense
patience, support and constructive criticisms during the writing of this chapter. I was supported by grants from the American Foundation for AIDS Research and the National Institutes of Health for my research studies cited in this chapter.
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189. Braselmann S, Graninger P, Busslinger M. A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins. Proc Natl Acad Sci USA 1993; 90(5):16571661.
190. Wang Y, OMalley BW, Jr., Tsai SY et al. A regulatory system for use in gene transfer. Proc Natl
Acad Sci USA 1994; 91(17):8180-8184.
191. Whelan J, Miller N. Generation of estrogen receptor mutants with altered ligand specificity for use
in establishing a regulatable gene expression system. J Steroid Biochem Mol Biol 1996; 58(1):3-12.
192. Bushman FD. Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences. Proc Natl Acad Sci USA 1994; 91(20):9233-9237.
193. Goulaouic H, Chow SA. Directed integration of viral DNA mediated by fusion proteins consisting
of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein. J Virol 1996;
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194. Re F, Luban J. HIV-1 Vpr: G2 cell cycle arrest, macrophages and nuclear transport. Prog Cell
Cycle Res 1997; 3:21-27.
195. Krausslich HG, Ochsenbauer C, Traenckner AM et al. Analysis of protein expression and virus-like
particle formation in mammalian cell lines stably expressing HIV-1 gag and env gene products with
or without active HIV proteinase. Virology 1993; 192(2):605-617.
196. Shoeman RL Honer B, Stoller TJ et al. Cleavage of the intermediate filament subunit protein
vimentin by HIV-1 protease: utilization of a novel cleavage site and identification of higher order
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197. Shoeman RL, Mothes , Honer B et al. Effect of human immunodeficiency virustype 1 protease on
the intermediate filament subunit protein vimentin: cleavage, in vitro assembly and altered distribution of filaments in vivo following micrinjection of protease. Acta Histochem Suppl 1991; 41:129141.
198. Corbeau P, Kraus G, Wong-Staal F. Efficient gene transfer by a human immunodeficiency virus
type 1 (HIV- 1)-derived vector utilizing a stable HIV packaging cell line. Proc Natl Acad Sci USA
1996; 93(24):14070-14075.
199. Corbeau P, Kraus G, Wong-Staal F. Transduction of human macrophages using a stable HIV-1/
HIV-2-derived gene delivery system. Gene Ther 1998; 5(1):99-104.
200. Kafri T, van Praag H, Ouyang L et al. A packaging cell line for lentivirus vectors. J Virol 1999;
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201. Xu K, Ma H, McCown TJ et al. Geneation of a stable cell line producing high-titer self-inactivating lentiviral vectors. Mol Ther 2001; 3(1):97-104.
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202. Howcroft TK, Strebel K, Martin MA et a. Reprssion of MHC class I gene promoter activity by
two-exon Tat of HIV. Science 1993; 260(5112):1320-1322.
203. Kanazawa S, Okamoto T, Peterlin BM. Tat competes with CIITA for the binding to P-TEFb and
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204. An DS, Wersto RP, Agricola BA et al. Marking and gene expression by a lentivirus vector in
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CHAPTER 6
Abstract
96
Pathogenesis
In domestic cats, FIV pathogenesis is primarily characterized by a progressive incapacitation of the immune system. Functional immunodeficiency begins early in the acute phase of
infection, as evidenced by a defect in T lymphocyte helper activity prior to any quantitative
defect in the CD4 positive T cell population.40 Unlike HIV-1, FIV does not exclusively infect
FIV Vectors
97
CD4 positive cells; therefore, the direct cytotoxic effect of FIV infection observed in vitro41
cannot in itself account for the rapid decline in CD4 cell counts in vivo. Similar to HIV,
mechanisms of CD4 cell depletion such as indirect triggering of apoptosis in uninfected cells42,43
and impaired T-cell regeneration due to premature involution of the thymus in juvenile cats44
are induced by FIV infection.
In addition, the altered cytokine network induced by FIV infection can also account for
immune dysfunction, notably increased expression of IFN-, TNF- and IL-10 mRNAs and
decreased expression of type 1 cytokines IL-2 and IL-12 mRNAs45,46 Therefore. similar to
HIV-1 infection,47 the apparent decline in Th1 immunity combined with an increase in IL-10
expression seems to constitute a key factor in FIV-induced immunodeficiency.
Another clinical hallmark of FIV pathogenesis is the development of neurological abnormalities in some infected animals,1 similar to what is seen in HIV-1 infection.48 The first direct
evidence of FIV neurotropism was reported by Dow et al (1990) who were able to isolate FIV
from cultures of neural cells from different areas of infected animals central nervous system
(CNS).24 Anatomically, FIV-infected animals show signs of neural tissue injury with loss of
neurons in the frontal cortex49,50 and gliosis of gray and white matter.49,51 Neurological symptoms include altered behavior and are correlated to signs of dysfunctional physiology of the
central and peripheral nervous systems.52-57 Like HIV-1, FIV is believed to penetrate the CNS
via infected cells capable of crossing the blood-brain barrier (BBB), although the disruption of
the BBB observed in some animals during acute infection may also allow penetration of cellfree FIV particles in the CNS.55 To date there is no evidence of direct neuronal infection by
FIV, although, astrocytes and microglia have been found to be infected in vivo, however at low
levels.51,58 FIVs neurotoxicity mechanisms are poorly understood, however FIV infection inhibits astrocytes uptake of glutamate, whose increased concentration in the microenvironment
is responsible for neuron damage.56,59,60 FIV infection also induces abnormal microglial expression of cytokines, notably TNF-, which is believed to play an important role in FIV
neuropathogenesis, as it is the case for HIV-1 and SIV infections.61,62
98
FIV Vaccines
In the past decade, many types of vaccine preparations consisting of either inactivated
virus, inactivated FIV infected cells, subunit recombinant proteins, peptides, plasmid DNA or
viral vectors encoding FIV epitopes have been tested in cats for their potential protective effect
against FIV infection (for review see ref. 78).79-81 Inactivated whole virus preparations and
infected cells have been shown to successfully protect animals from infection with homologous
or closely related isolates.82-84 Interestingly, subunit vaccines administered either as recombinant proteins alone or via immuno-stimulating complexes have shown little or no protective
activity, and in some cases were associated with increased susceptibility to subsequent challenge
infection.85-87 More recently, DNA immunization with a live attenuated FIV showed protection from infection with wild-type homologous virus.88
When achieved, long-term protection appears to be mediated by CTLs,89,90 which can be
transferred to nave animals and confer protection to homologous challenge in a MHC restricted manner.91 While high levels of virus-neutralizing antibodies in vaccinated animals do
not correlate with enhanced protection from challenge by homologous strains, they do seem to
be important for relative protection against infection by heterologous strains.92 Overall, FIV
vaccination studies parallel those derived from nonhuman primate lentivirus studies in that
broadly cross-neutralizing intersubtype protection constitutes a challenging goal. Still, recent
encouraging results suggest, that vaccination trials in the FIV/cat system could provide important information useful for the design of a candidate vaccine for HIV infection.92,93
Antiviral Drugs
Given the common structural and biochemical properties between HIV and FIV enzymatic proteins, drugs that inhibit HIV replication, such as reverse transcriptase (RT) and certain protease inhibitors, also inhibit FIV enzymes activity in vitro.94-98 In vivo, RT inhibitors
improve clinical manifestations of FIV infection, notably in young cats.99,100 Unfortunately,
the similarities between HIV and FIV extend in their capacity to develop drug resistance by
evolving escape mutants harboring amino acid substitution in the protein sequences of RT.101,102
FIV molecular clones were sequenced soon after the first description of the virus.103,104
Phylogenetic analyses have revealed that FIV is more closely related to non-primate lentiviruses
than to the HIV/SIV subgroup,103 although as described above, pathologies associated with
FIV infection are more reminiscent of those of HIV and SIV than of those of the equine
infectious anemia virus (EIAV), visna-maedi virus (VMV) and caprine arthritis-encephalitis
virus (CAEV). The 9.2 kilobase genome of FIV harbors typical retroviral/lentiviral structures
(Fig. 1). As for all retroviruses, the FIV proviral genome is flanked by two long terminal repeats
(LTRs) generated by the duplication of the genomic terminal sequences during reverse transcription. The coding sequences include the gag, pol and env retroviral genes along with various
other open reading frames (ORFs), whose expression relies on alternative splicing mechanism
(Fig. 2). Most of these ORFs encode accessory/regulatory proteins that have been characterized
and have revealed striking structural and/or functional similarities with proteins encoded by
other lentiviruses more or less distant in evolution.
FIV Vectors
99
Fig. 1. FIV proviral genome organization relative to other lentiviruses. The relative positions of characterized open reading frames and Rev responsive elements within each genome are shown.
revealed the AP-1, AP-4, ATF and C/EBP elements capacity to interact with cellular proteins,
although their binding properties appear to vary in different cell types.106-108 AP-1, ATF and
C/EBP sites are important for basal promoter activity in various in vitro systems.105,106,108,109
The ATF binding site is responsible for FIV LTR increased activity following stimulation of
the protein kinase A signal transduction pathway and the AP-1 site confers responsiveness of
the viral promoter to phorbol-esters and to lymphoid cell activation signals.105,110 Deleting the
AP-1 site does not impair FIV replication in feline PBMCs or macrophages,110,111 however
virus replication and pathogenesis are affected in vivo.112 On the other hand, deleting the ATF1 binding site alone or in combination with an AP-1 deletion is detrimental to virus replication
in PBMCs and macrophages111 consistent with the transactivation studies by transfection.106
Furthermore, deleting the entire enhancer region virtually abolishes virus replication in otherwise permissive cells.
Various studies conducted to determine whether the FIV genome encoded its own
transactivator had led to conflicting results about the orf2 gene products ability to activate FIV
LTR driven expression.30,105,108 However, de Parseval et al recently have brought strong evidence that Orf-2 was capable of transactivating the basal activity of FIV enhancer/promoter by
up to 20-fold depending on the cell type.113 The mechanism of this transactivation observed in
feline as well as in human fibroblasts has yet to be defined. While no direct binding of Orf2
protein to the FIV LTR has been observed, deletions of the AP-1, C/EBP tandem or ATF sites
alone or the deletion of the region encompassing AP-1 through ATF decreased or abolished the
transactivating effect of Orf2 respectively, suggesting the involvement of these regions in Orf2mediated LTR transactivation.
100
Fig. 2. FIV genome expression pattern. Functional splice sites generating alternately spliced transcripts are
shown. The size of transcripts and the nature of encoded genes are indicated. p15rev was recently identified
by de Pareseval et al and was shown to exert partial Rev activity in feline cells.113
FIV Vectors
101
Protease (PR)
The 13 kDa FIV PR is responsible for processing the Gag and Gag-Pol polyproteins into
their mature products. Although FIV and HIV protease share many structural features
(homodimeric nature and similar quaternary structure120), they demonstrate distinct specificities for substrates and inhibitors.98,121 Comparative studies between primate lentivirus proteases and the FIV protease, combined with mutagenesis analyses, have led to the identification
of common amino acid residues involved in the recognition of protease ligandsimportant
for the development of protease inhibitors that are effective against different virus species and
against resistant mutants.97,122,123
Integrase (IN)
The 32 kDa FIV IN is located at the C-terminus of the Gag-Pol polyprotein. FIV IN
shows catalytic activities typical of integrase enzymes in vitro, i.e. site-specific cleavage of FIV
viral DNA ends, DNA strand-transfer and disintegration.130 Functional studies suggest that
FIV IN is a multimer, like other retroviral integrases.131 The FIV IN functional domains include the N-terminal domain, which contains a His-Cys zinc-finger motif involved in DNA
interaction, and the central domain that contains the sequence D-X39-58-D-X35-E specific of
integrase catalytic sites (for review see ref. 132). Additionally, the FIV IN C-terminus region
also plays an important part in the enzymes interaction with its DNA substrate.131 An FIV
molecular clone harboring a 121 amino-acid deletion in the C-terminus of the protein transfected into CrFK cells produced immature noninfectious particles that contained high levels of
unprocessed Gag precursor and no detectable RT activity, indicating a role for FIV integrase
protein in the late stages of FIV replication133 as suggested for HIV-1 integrase.134
102
Envelope
FIV Env is encoded in a singly spliced mRNA of 4.4 kb (Fig. 2). FIV Env precursor
processing into a glycoprotein arranged in a non-covalent association between SU (gp120) and
TM (gp40) subunits results from post-translational modifications common to all retroviral
envelopes. However, like CAEV Env, FIV env undergoes an additional proteolytic processing
that is responsible for the cleavage of a 20 kDa polypeptide present at the N-terminus of the
SU domain142 and which includes the 80 N-terminal residues of the rev first exon. Little is
known about this 20 kDa fragment structure or about its subcellular localization. However,
FIV Vectors
103
Vahlenkamp et al recently reported that a FIV mutant partially deleted in the 20 kDa polypeptide
region exhibited dramatically decreased replication levels in feline primary astrocytes and in feline
astrocyte-derived cell lines, while replication of the same mutant was unaffected in other cell types
including PBMCs and CrFK cells.143 Whether the deletion impaired the viral envelope function
in astrocytes at an early or late stage of viral replication remains to be determined.
Env is the most divergent structural protein of FIV.144 Similar to HIV, FIV Env is structured in conserved and variable regions.145,146 Nine variable regions are scattered throughout
the FIV Env molecule: two of them (V1, V2) are located in the 20 kDa domain, four (V3-V6)
in the SU domain and three (V7-V9) in the TM coding region. Domains V1 through V5 are
classified as hypervariable;146 V3 and V4 domains as well as the ectodomain of TM of FIV Env
are implicated in FIV tropism.26-28 As with HIV, a principal neutralization domain and an
immunodominant domain have been identified in FIV V3 and in the ectodomain of TM
respectively.147 FIV Env also holds virus tropism determinants within its sequence26-29 and
although HIV and FIV interact with CXCR4 to enter target cells, they share little homology in
their envelope protein sequences. Interestingly, Serres148 recently reported structural and physical
analogies between the trimeric models of TM ectodomains of HIV, SIV and FIV and the IL-2
molecule of their respective species. These findings suggest that three moreorless distantly related immunodeficiency viruses have evolved a common structural feature to specifically interact with their host target cells. Further studies are needed to determine to what extent this
contributes to FIV, HIV and SIV pathogenesis.
104
in 1998,157 was followed by the optimization of FIV vector technology including the first in
vivo studies in 1999.158 Curran et al describe a potential benefit of FIV vectors, suggesting that
FIV does not efficiently cross-package HIV genomic RNA. If confirmed, this trait may be
desirable for the gene therapy of HIV-infected subjects.159 Further in vivo studies established
FIV vectors as an efficient gene therapy vector. Transduction of the primate and murine central
nervous system160-162 and rabbit airway epithelium have resulted in longterm expression at
therapeutic levels. Although these initial studies were carried out with VSV-G pseudotyped
FIV vectors, FIV vectors have also been successfully pseudotyped with the amphotropic envelope.
In fact, MLV, HIV-1 and FIV-based vectors carrying the amphotropic envelope have a prolonged half-life in human serum compared to these vectors pseudotyped with VSV-G,163 a
particularly important finding for those applications requiring direct injection into blood vessels.
When FIV vectors were developed, relatively little was known about the sequence requirements for efficient packaging of the FIV RNA genome or the activity of certain cis elements in
human cells. By analogy with other lentiviruses, the location of the putative packaging signal
for FIV was suspected downstream of the 5LTR, possibly extending into gag coding sequences.
More was known about the activity of the FIV promoter in human cells: reports described that
feline-specific tropism of FIV is governed by low transcriptional activity of the FIV LTR in
human cells as well as its envelope.105,141,164-167 More recent data indicate that wild-type FIV
can penetrate into certain human cells via the interaction of its envelope with human CXCR4.
While there are reports of FIV infection of human cells without spreading of the virus in
culture,164,167,168 low levels of infection of human primary PBMCs are observed albeit without
evidence for FIV integration into the genome. Currently, we still have little understanding as to
what receptor(s) other than CXCR4 may be involved in FIV wild-type infection, and because
of that we suggest the state-of-the-art safety measures developed for other lentiviral vectors will
also be incorporated into FIV vectors despite any evidence for disease in humans caused by
exposure to FIV.
Low FIV LTR activity in human cells along with other potential roadblocks caused researchers to focus on designing an FIV vector system to overcome possible restrictions inherent
to the FIV virus. The first steps in FIV vector development addressed basic questions such as i)
the ability to accommodate heterologous human-tropic envelopes; ii) FIV LTR activity in human cells; iii) the packaging signals size and location; iv) the need for RNA export elements in
the transfer vector and packaging constructs; and v) the influence of accessory proteins on titer
and transduction efficiency.157-159 All presently described FIV vector systems have been derived from FIV-34TF10, a variant of the Petaluma strain,12,103,104 which productively infects
feline kidney and neuronal cells but not PBLs or macrophages. Also, the FIV-34TF10 molecular clone is readily available from the NIH AIDS Research and Reference Reagent Program (Cat#
1236). To generate FIV vectors, the plasmid components of the recombinant vector system
were transfected into the human 293T cell line.169 This same highly transfectable producer
line has been used for the transient production of MLV and lentiviral vectors and allows rapid,
high-titer viral particle production. Using human cell lines for FIV vector production reduces
possible problems from recombination of FIV vector components with endogenous feline
retroviral sequences or potential infectious agents associated with less well-characterized feline
producer lines. In addition, production in a human line avoids destruction of viral particles by
human serum complement as documented for MLV vectors.170-173
Since clinical trials in 1990,174 experience gathered with MLV-based retroviral vectors
suggests that, in addition to overall quality and titer, there is another critical issue in vector
production: safety regarding the generation of replication competent virus. The challenge is
generating FIV constructs with minimal sequence overlap in an effort to reduce the potential
for homologous recombination while maintaining the high-titer capacity. To address these
safety issues, FIV vector development followed the general design of state-of-the-art lentiviral
FIV Vectors
105
vector technology, i.e., separating the cis-acting sequences involved in the transfer of the viral
genome to target cells from the sequences encoding the trans-acting viral proteins. This splitgenome strategy results in separate expression cassettes for the FIV transfer vector, the packaging plasmid and the envelope plasmid. Because of this, currently described FIV vector systems
consist of three or four components depending on whether FIV or HIV Rev protein is expressed
from its own plasmid (Fig. 3). The resulting recombinant FIV vectors are replication-incompetent
and limited to a single round of the infection process in the target cell without spreading.
106
Fig. 3. Schematic presentation of the plasmid components for the FIV-based gene delivery system. Represented are the FIV transfer vector (A), the FIV packaging plasmid (B), and a plasmid coding for a heterologous viral envelope (C). The FIV packaging plasmid can be Rev/RRE independent using heterologous
export elements such as the CTE, or the packaging functions use an Rev/RRE export element with the Rev
protein being driven by a separate plasmid (B). Prom.: promoter.
and 3 end, respectively, replaces both FIV LTRs and the putative packaging signal. In addition,
the plasmid also provides an RNA export element in the form of the original FIV Rev/RRE or
the heterologous CTE or HIV Rev/RRE sequences. First-generation FIV vectors retained the
FIV-34TF10-derived accessory proteins Vif (functional) and Orf2 with the premature stop
codon (nonfunctional), while later generations of FIV vectors158,159describe packaging plasmids without either of the accessory proteins. A prototype of the FIV packaging plasmid with
a Rev/RRE export element included in the same packaging plasmid (three plasmid system) or
provided on a separate plasmid (four plasmid system) is shown in Figure 3B.
Future efforts to improve the safety and expression level of the FIV core proteins may
include the use of degenerate code previously described for retroviral vectors. Degenerate codons
will not only eliminate sequence homology in the 5 area of the packaging plasmid with the
packaging signal of the FIV vector but may also increase expression levels of the Gag-Pol proteins and generate an mRNA independent of any export element as described for the expression of HIV core proteins.177,193-196
Envelope Plasmid
The flexibility to incorporate non-FIV envelope glycoprotein into the FIV viral particle
provides an exciting opportunity for cell and tissue targeting using the natural or modified
tropism of the heterologous viral envelope. So far, the pantropic VSV-G197 and the 4070 Aderived amphotropic envelopes198,199 have efficiently pseudotyped FIV vectors.157-159,163 A
heterologous promoter and poly A signal regulate the expression of the envelope of choice. A
prototype of the envelope plasmid is shown in Figure 3C, and it is not surprising that it does
not differ in general design from envelope cassettes described for other lentiviral vectors.
Compared to VSV-G pseudotyped FIV vectors, pseudotyping with the amphotropic envelope results in an approximately five- to tenfold lower titer in unconcentrated supernatant.
This is also true for MLV- or HIV-based vectors (unpublished data). The higher titers observed
for VSV-G pseudotyped vectors may be a result of increased transduction efficiencies rather
FIV Vectors
107
Fig. 4. In vivo transduction by a VSV-G pseudotyped FIV vector of (A) hamster muscle;158 (B) Purkinje
cells in the murine cerebellar lobule (2x105 cfu, day 21), courtesy of Dr. Beverly Davidson; and (C) rabbit
airway epithelium transduced from the apical side.214 Various cell types of the airway epithelium including
ciliated and basal cells are transduced (indicated by arrows).
than higher recombinant viral particle output. Possible explanations for increased transduction
include the higher prevalence of VSV-G receptors compared to amphotropic receptors on target cells, different mechanisms for viral particle uptake and/or increased half-life of VSV-G
pseudotyped particles in vitro. Incorporating VSV-G into the recombinant viral particle results
in increased resistance to mechanical stressesultracentrifugation, for example.200 This property is not observed in amphotropic vectors and may contribute to a reduced half-life in culture
media and increased losses of infectivity during vector concentration. Like many animal viruses that bind to specific receptors on the plasma membrane of cells, the amphotropic envelope receptor Pit-2 is a phosphate transporter that interacts with the amphotropic envelope in
a very specific manner.198,199,201,202 In contrast, the receptor for VSV-G is a rather nonspecific,
ubiquitous phospholipid molecule203-205 present in all cell membranes, which results in such a
broad host range that it efficiently transduces almost all species of vertebrate and insect
cells.200,206,207 Furthermore, viruses with VSV-G envelopes enter the target cells through general endocytosis via clathrin-coated pits and pH-induced endosomal fusion208 while viruses
with amphotropic envelopes fuse directly at the cell membrane after binding to the receptor.
The exact mechanism(s) of reduced titer of amphotropic compared to VSV-G tropic vectors
remains to be elucidated.
The general pseudotyping capability of the FIV particle is not yet explored in great detail,
but the high titer VSV-G and amphotropic FIV vectors hold promise for other envelopes.
Interestingly, the Gibbon Ape Leukemia Virus (GALV) envelope efficiently pseudotypes MLV
but not HIV vectors, and our observations show that the GALV envelope does not pseudotype
FIV vectors either (unpublished data). However, a recent report describes the successful
pseudotyping of HIV vectors with the GALV envelope after exchange of the cytoplasmic tail of
GALV with that of the MLV envelope in an effort to mimic the amphotropic envelope.209
Although HIV vector titers with the GALV hybrid envelope are low, and the most commonly
used envelope for lentiviral vectors is VSV-G, alternative envelopes and envelope hybrids widen
the opportunity to transduce specific cell types and enhance the FIV vectors utility for a range
of applications where more restricted tropism is beneficial.
Despite the higher titer of VSV-G pseudotyped FIV vectors, we propose that the
amphotropic envelope may have advantages over VSV-G, in particular for developing clinicalgrade FIV vectors. In addition to the excellent safety profile of the amphotropic envelope
108
demonstrated by the safe administration of amphotropic MLV vectors to over 1,000 patients210
further benefits are i) lack of toxicity caused by the fusogenic property of VSV-G; ii) resistance
to human serum inactivation; iii) the possibility of generating stable packaging and producer lines
without the need for regulated vector production and; iv) reduced pseudotransduction.211,212
FIV Vectors
109
Accessory Proteins
The simple FIV genome, with only two accessory proteins (Vif, Orf2) and one regulatory
(Rev) protein, facilitates analyzing their importance for the FIV vector system. To study possible effects of vif and orf2 coding regions and/or their proteins, packaging plasmids were generated containing either vif or orf2 alone, both genes or none. Since FIV-34TF10 is naturally
dysfunctional in Orf2 expression,104 its orf2 coding region was substituted with that of the
fully functional orf2 of the molecular FIV strain FIV14.158 Resulting FIV vectors were used to
analyze the titer and transduction efficiency of various proliferating and nonproliferating cells.
Transduction efficiencies of vectors prepared without the FIV vif and orf2 genes did not differ
substantially from those of vectors prepared with accessory gene expression in either dividing
or nondividing cells (ref. 158 and Table 2). This finding is comparable to the situation in HIV
vectors where efficient transduction does not require any of the accessory proteins except for
one reported case of diminished transduction of liver after in vivo injection with recombinant
HIV-1 vectors lacking the accessory Vpr and Vpu proteins.215 Interestingly, studies also report
efficient transduction of primary human hepatocytes by FIV vectors irrespective of the presence or absence of sequences coding for FIV accessory proteins.159
Based on the function of FIV Vif and the fact that HIV Vif(-) vectors do not show diminished titer production or transduction efficiency in vivo,216 we anticipated that there may not
be a need for FIV accessory proteins for efficient FIV vector performance either. The
transactivating activity of Orf2 becomes superfluous in the context of FIV vectors with a hybrid LTR, suggesting that retaining Orf2 in FIV vectors may not have an advantage over FIV
Orf2 vectors. Altogether it may not be surprising that FIV vectors deleted in accessory proteins
show undiminished titer and transduction efficiency although it cannot be ruled out that accessory proteins or their sequences may play a role in the transducibility of certain species or
tissues that have not yet been examined.
110
FIV/gal, VSV-G
FIV/gal, Ampho
Mean Titera (CFU/ml of virus stock) SD
HeLa
HT-1080
293T
CrFK
3T3
FIV vectors coding for -galactosidase were pseudotyped with the envelope from Vesicular Stomatitis
Virus (VSV-G) or the amphotropic envelope from 4070A (Ampho). Vectors were concentrated and
virus stocks titered on a variety of cell lines. a Each titer value represents the average of duplicate titers
determined from 2-8 titer values scored at different dilutions each. Titer is expressed as LacZ colonyforming units per milliliter of virus stock.
Export Elements
To understand the requirement for the FIV vector system regarding RNA export elements, FIVs natural RNA export system (FIV Rev/RRE), as well as substitutions with heterologous export elements were analyzed in the context of the FIV vector and packaging plasmid.158,159 The natural location of the FIV RRE at the 3 end of the genome facilitated the
design of FIV vectors and packaging plasmids since the RRE site could be retained in its original position as shown in early generation vectors.157-159 An alternative location for the FIV
RRE in the vector construct upstream of the internal expression cassette of the transgene resulted in slightly increased titers.158 Heterologous export systems such as the Rev/RRE system
from HIV-1 achieved 1.5- to 5-fold increased titers,158,159 whereas the CTE export element
gave titers comparable to the FIV Rev/RRE system as long as the CTE was located less than
250 bp upstream of the 3FIV LTR.159 In the absence of any export system in the FIV vector
and packaging construct, the titer dropped by as much as five logs. Lack of the FIV RRE in the
FIV vector alone while maintaining the FIV Rev/RRE system in the packaging construct reduced the titer by approximately 1-1.5 logs.158 In general, both the FIV vector and packaging
plasmid require an export element for high titer vector production, although the nature and
location of the export element is flexible and certain heterologous export systems may help to
increase titers relative to FIVs own export system. Future modifications of the packaging plasmid may include the use of degenerate codons to increase expression levels and gain independence from any export element as described for HIV Gag-Pol.194
Safety Modifications
The major safety concern for lentiviral vectors is the emergence of replication-competent
virus, which usually arises from homologous and to a lesser extent nonhomologous recombination.217,218 In an effort to reduce the substrate for homologous recombination, the sequence
homology between FIV vector components is minimized. Corresponding safety modifications
FIV Vectors
111
Packaging Construct
Vector Construct
pCFIV
pVETLC
pCFIVorf2
pVETLC
pCFIVvif
pVETLC
pCFIVorf2vif
pVETLC
pMLVgagpol
pMLVCMV
a Results are from a representative experiment, with each value representing the average transduction
efficiency of three replicate vector preparations done in triplicate. Transduction efficiency is expressed
as LacZ-forming units per milliliter of virus stock. Virus stock was used to transduce dividing and
nondividing human skin fibroblasts at an MOI of 2.0. The transduction efficiency of vector stocks
prepared from pCFIV and pVETLC was arbitrarily given a value of 100%, to which the transduction
efficiencies of the other vector stocks were compared. FIV packaging constructs code either for Vif and
Orf2 (pCFIV), Vif only (pCFIVorf2), Orf2 only (pCFIVvif) or neither accessory protein (pCFIVorf2vif).
The FIV and MLV vectors pVETLC and pMLVCMV, respectively, code for -galactosidase.
of the FIV vector system include: i) replacement of the FIV LTRs with heterologous promoters
and poly A signal in the packaging plasmid; ii) separation of viral structural and enzymatic
genes into at least two expression cassettes, gag/pol and a heterologous env; iii) reduction of
sequence homology between the individual retroviral components; and iv) the use of human
producer cells instead of feline cells. All presently described FIV vectors were generated in
human cells and follow the design of the split-genome approach with minimal sequence overlap.157-159 To further separate the FIV components, expression of the FIV Rev protein was
driven by its own expression plasmid, resulting in a four-plasmid transfection system for FIV
vector production (Fig. 3).158,159 Alternatively, heterologous export elements such as the CTE
eliminated the need for FIV Rev and RRE sequences altogether;159 however, one area of sequence overlap currently remains between the 3 area of the putative packaging signal in the
FIV vector and the 5 part of the packaging plasmid.
Following these general guidelines, minimal FIV packaging constructs were designed and
the production of accessory proteins dispensable for vector production and infectivity was
eliminated. A minimal packaging construct with only 6 bp of noncoding sequence upstream of
the major splice donor (MSD) site while deleting the 17 bp normally located between the
MSD and the gag start codon is as functional as a packaging plasmid with additional 100 bp
upstream of the MSD and the 17 original bp between MSD and gag start remaining. Downstream of the FIV gag/pol coding region, deletions in the envelope and accessory protein
112
sequence of various sizes were introduced to disable the generation of the Env and/or accessory
proteins. The most advanced packaging constructs contain heterologous HIV Rev/RRE or
CTE export elements.158,159
FIV vector constructs are generally deleted in all FIV structural and enzymatic genes.
However, due to its dual function as a part of the putative packaging signal as well as the 5
coding part of gag, partial sequence of the gag-coding region is retained. The shortest packaging
signal reported in an FIV vector without titer loss includes the 270 bp between the 5 LTR and
the gag codon plus the first 350 bp of the gag coding region.158 These first definitions of the
putative packaging signal await further, more detailed analysis of the minimal requirements for
maximal packaging. Additional safety features include the introduction of a stop codon around
300 bp downstream of gag start to avoid production of a Gag-Pol multiprotein in case recombination between the FIV vector and packaging plasmid occurs.157 The next generation vectors
eliminated most of the remaining envelope and FIV Rev/RRE sequences present in the first
generation FIV vectors.157-159 Table 3 summarizes in more detail the molecular structure of the
currently published FIV vector systems, each describing the most advanced versions of the FIV
gene delivery system.
Extensive testing for replication-competent virus generated during FIV vector production
has not yet been described, but our efforts to monitor FIV p27 (capsid) expression levels in
human and feline target cells exposed to high titer FIV vectors and passaged for a period of six
weeks did not result in any p27 expression levels above background (unpublished data). Analogous to the RCR testing of primate lentiviral vectors, additional RCR assays for FIV vectors
including quantitative PCR need to be developed to assure this nonprimate lentiviral gene
delivery systems safety.
FIV Vectors
113
gag-coding sequences of
interrupted by stop codon
Heterologous export element
FIV packaging plasmid
Remaining FIV sequence upstream
of gag/pol start (bp)
Sequence between MSD and
start of gag eliminated
Heterologous export elements
Rev coded on a separate
expression plasmid
Sequence coding for accessory
proteins deleted
Poeschla et al,
1998157
Johnston et al,
1999 158
Curran et al,
2000159
1,250 bp
350 bp
830b bp
120 bp
-
10 bp
+
120 bp
-
+
+
+
+
a Compared were the most advanced vector components in the respective publications.
b Reported length of the 3rd generation transfer vectors. However, earlier generation transfer vectors
Extensive transgene expression and duration in a variety of tissues and species is seen in the
absence of inflammatory responses.214 Particularly promising for a gene therapy approach to
cystic fibrosis is the stable expression of a transgene in 5-10% of airway epithelium transduced
from the apical side,214 since that level of transduction is in a range considered to be
therapeutic.222 Detailed analysis of the cell types transduced by FIV vectors following injection
into mouse cerebrum revealed that Purkinje cells among others were successfully transduced.160
This suggests that FIV vectors may be applied for diseases affecting the cerebellum such as
spinocerebellar ataxias (SCA). Stable expression of -glucuronidase in a -gluc-deficient mouse
model after FIV/-gluc vector treatment resolved characteristic disease symptoms of
mucopolysaccharidosis VII.161 These data further validate FIV vectors for gene therapy applications of the mammalian central nervous system. Efficient gene transfer to the retina and liver
was observed as well (personal communication, Drs. Beverly Davidson and Paul McCray).
Figure 4 shows some examples of transduced organs and cells after direct injection of FIV
vectors. For in vivo studies, transiently generated FIV vectors were concentrated to achieve
titers ranging from 1x107-1x109 cfu/ml using a variety of methods including ultrafiltration,
centrifugation, ion exchange chromatography and PEG-precipitation. Table 4 shows an example for FIV vector concentration using PEG precipitation followed by centrifugation.
Final titer and general quality of the preparation is an important factor ultimately influencing the in vivo efficacy of retroviral/lentiviral vectors. Vector preparations can vary greatly
in their quality, especially concerning the ratio of infectious versus noninfectious vector as well
114
Amount (ml)
Titer (TU/ml)
Concentration
(x-fold) recovery
% step
recovery
Crude
supernatant
5,000
3.1 x 105
Clarified
5,000
6.8 x 105
221
PEG concentrate
200
1.1 x 107
25
62.9
Centrifuged, final
2.0 x 108
1667
27.9
% overall
38.8
Representative concentration procedure for FIV vectors using PEG precipitation followed by pelleting.
Unconcentrated viral supernatant is clarified using a filtration step (0.45 um) followed by polyethylene
glycol precipitation (10% PEG final) for at least 4 hours at 4oC followed by pelleting and resuspension
in a smaller volume. FIV vectors were then further concentrated to a smaller volume by a final
centrifugation step. FIV vectors were pseudotyped with VSV-G, coded for erythropoietin and titers
were determined by quantitative PCR.
as protein and other contaminants, for example FBS. A detailed description on the production
of high quality MLV-based vector is noted elsewhere,223 however, these observations most likely
apply to FIV vectors as well. The development of stable FIV packaging and producer cell lines
is expected to improve the quality and reproducibility of FIV vector preparations. Extensive
screening of producer lines for high titer and general scalable growth characteristics usually
identifies a candidate with a high ratio of infectious to noninfectious particles. High-titer producer cell lines will facilitate a commercially feasible manufacturing process that will in turn
deliver good quality vector at the clinically relevant titers required for therapeutic responses.
Will lentiviral vector systems derived from various species perform in a similar fashion in primates? Some lentiviruses naturally favor certain tissues and cell types, a feature often defined by
the envelope component. However, as in the case of FIV-34TF10 where deletion of functional
orf2 results in modified cell tropism, it is conceivable that differences in the activity of ciselements and non-envelope proteins such as the integrase or reverse transcriptase modulate the
overall efficiency of a given lentiviral vector in primates. Comparing different lentiviral systems
is difficult since inequalities in vector material flaws most comparisons. To assess relative efficiencies of lentiviral vectors from different species, all components going into the final vector
preparation must be generated and treated in the same manner, starting with the quality of the
plasmids, the nature of the vector components (i.e. export elements and promoters), vector
production, purification and titration.
Apart from using FIV vectors for human gene therapy, the technology is beneficial for
anyone who needs to efficiently introduce transgenes into any type target cell for further study,
or to generate stable cell lines. FIV vector production is both simple and fast, and researchers
may use FIV vectors to study basic FIV virology. In summary, primary results from in vitro and
in vivo studies of FIV vectors indicate that this nonprimate gene delivery system is highly
efficient and holds promise for many human gene therapy applications.
FIV Vectors
115
Acknowledgements
This work was supported in part by the National Institute of Allergy and Infectious Diseases grant AI45992.
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CHAPTER 7
Abstract
Introduction
A general property of lentiviruses is the ability to integrate their genomes into the chromosomal DNA of non-dividing infected cells. This property, which is not shared by simple
retroviruses such as murine leukemia virus or avian leukosis/sarcoma virus, is the main rationale for developing lentiviruses for gene transfer applications.1 At present vector systems have
been derived from eight different lentiviruses including HIV-1, HIV-2, SIV, FIV, EIAV, CAEV,
visna virus, and JDV. Earlier Chapters in this volume describe the development and properties
of the first four of these vector systems, while this Chapter describes the construction and
properties of the latter four gene transfer systems.
127
Fig. 1. Genomic organization of selected lentiviruses. Shaded boxes indicate gag, pol and env genes. Solid
boxes indicate accessory genes.
128
The genetic organization for these lentiviruses is shown in Figure 1. The lentiviral genome
contains gag, pol, and env genes that encode the structural and enzymatic proteins that make up
infectious viral particles. These viruses also contain tat and rev accessory genes that encode
proteins that regulate gene expression. While these viruses typically have multiple exons encoding tat and rev some differences can be noted for the placement of the coding exons for these
genes. For example, the first exon of EIAV tat is located 5' to the gag gene before the major
splice donor site. An unusual CUG codon is used to initiate EIAV tat translation.2 An noteworthy feature in the organization of JDV is that both env and the second exon encoding rev
extend into the 3' LTR sequence,3 similar to the nef gene of HIV-1 and other primate lentiviruses.
EIAV, CAEV, visna, and JDV all contain a third accessory gene. For CAEV, visna, and
JDV this gene appears to be analogous to HIV vif. The placement of vif for each is similar and
overlaps the 3' end of pol. Based on comparisons of the predicted protein, it is thought that the
non-primate Vif proteins are functionally analogous to HIV Vif.
EIAV S2 Gene
EIAV differs from the other lentiviruses in lacking a gene encoding a recognizable Vif
protein. Instead EIAV contains a gene called S2, which is located in the pol-env intergenic
region. The S2 protein is expressed from a singly spliced tricistronic mRNA, also potentially
coding for Tat and Env.2,4 Translation of this mRNA to make S2 uses an AUG codon 10 nt
downstream of the open reading frame for Tat and 26 nt upstream of the env AUG in an
alternate reading frame. S2 encodes a 65 amino acid protein and has several potential functional motifs that are conserved among various isolates and remain conserved during virus
genome evolution within an infected animal.5 These sequences conform to nucleoporin (GLFP),
SH3 domain binding (PXXP) and nuclear localization (RRKQETKK) motifs. In vitro synthesized S2 has been shown to react with anti-serum from EIAV infected horses,2 so S2 must be
expressed in vivo. The role of S2 in virus replication remains unclear. Mutagenesis of S2 in an
infectious, pathogenic molecular clone of EIAV had no affect on viral replication kinetics in
equine monocyte/macrophage cultures.6 However, mutation of S2 severely affected viral replication and virulence of EIAV in experimentally inoculated ponies.5
dUTPase
A common feature of non-primate lentiviruses is the presence of sequences encoding a
dUTPase protein. For FIV, EIAV, CAEV and visna virus the dUTPase is encoded by sequences
within the pol gene between sequences encoding RT and IN.7 As with RT and IN, dUTPase is
incorporated into viral particles. The bovine lentiviruses, BIV and JDV, lack dUTPase.3 Primate lentiviruses including HIV and SIV strains also lack a functional dUTPase, although
sequences related to dUTPase genes can be found within the env gene of HIV-1.8 Type B and
D retroviruses also contain sequences encoding dUTPase but these sequences are located in the
pro reading frame.9,10 Other viruses including herpesviruses11 and poxviruses12 have dUTPase
activity as do most, if not all, prokaryotic and eukaryotic organisms, including humans.13
Cellular dUTPase is part of an important biosynthetic pathway that catalyzes dUTP hydrolysis to dUMP and PPi with dUMP being converted to TTP by thymidylate synthase. The
dUTPase helps to maintain a low dUTP/TTP ratio (typically ~10-5) which is thought to be
important for minimizing the incorporation of uracil into DNA.14,15 Cellular dUTPase levels
appear to be regulated during the cell cycle and as a function of differentiation; as a norm
quiescent cells and terminally differentiated cells have lower levels of dUTPase than dividing
cells and non-differentiated cells.16-21 This provides a rationale for the presence of dUTPase in
lentiviruses whose target cells may be non-dividing. The role of viral dUTPase may be analogous to cellular dUTPase to prevent incorporation of uracil into reverse transcribed DNA.
129
Since uracil can base pair with guanosine this could lead to high G-to-A mutations if dU were
incorporated during first strand synthesis.
A number of studies support a role for dUTPase during lentiviral replication. Mutation of
dUTPase in FIV,22 EIAV,23,24 CAEV,25 and visna virus25 leads to delayed replication in nondividing primary macrophage cultures, but normal kinetics of replication in dividing primary
monocytes or permissive cell lines. In animals, dUTPase mutants show reduced viral loads in
EIAV infected ponies,24 visna virus infected sheep,26 attenuated severity of arthritic lesions in
CAEV infected goats,27 and decreased viral burden in some tissues of FIV infected cats.28
However, the neuropathogenic effect of dUTPase mutants was not different than wild-type
virus in FIV or visna virus infected animals.26,29
Mechanistic studies support the role of non-primate lentiviral dUTPase in preventing the
incorporation of dUTP into DNA during reverse transcription. G-to-A transition rates were
significantly elevated with dUTPase mutants compared to wild type virus during virus evolution in CAEV infected goats27 or FIV infected cats.28 Steagall et al investigated the molecular
defect causing the delay of replication in EIAV infected macrophage cultures where dUTPase
mutants replicate to 1-2% of wild type levels by 7 days post-infection.30 It was found that
reverse transcription was normal with full-length linear DNA evident 24 hr after infection.
Integration of dUTPase mutant DNA was slightly depressed (~2-fold) by 72 hr post-infection.
Transcription levels, however, were significantly depressed with the dUTPase mutant. By 72 hr
post-infection analysis, of RNA by Northern blotting showed about a 25-fold decrease in multiply spliced messages and an 85-fold to 300-fold decrease in full-length RNA in cells infected
with the dUTPase mutant compared to wild type virus. The decrease in RNA synthesis could
account for the decrease in virus production in these cultures. At present it is not clear how
RNA synthesis is depressed with the dUTPase mutant. Possibly, significant incorporation of
dU into the enhancer/promoter region could affect the interaction of transcription factors with
their binding elements. Using a PCR assay of uracil-DNA glycosylase treated DNA, Steagall et
al provided evidence that dU was incorporated into proviral DNA in macrophage cultures
infected with the EIAV dUTPase mutant.30
130
host and virus contribute to the eventual regulation of virus replication in inapparent carriers.
Some recent work has focussed on the viral determinants that influence the virulence. This
work has implicated the viral LTR, env, S2, and rev as having important roles in pathogenesis.5,34-36 The roles that these determinants have in disease progression are as yet incompletely
elucidated. The recent construction of pathogenic molecular clones of EIAV will be important
tools for dissecting viral elements that contribute to the disease process.34,35
Two groups have described lentiviral vector systems derived from EIAV.37,38 The design of
these systems is based on separation of cis-acting elements in the EIAV genome from sequences
encoding trans-acting proteins (Fig. 2). The viral cis-acting elements for replication and integration are contained in the gene transfer vector plasmid. Two other plasmids, the viral protein
expression cassette and an envelope expression plasmid, provide the genes encoding the viral
proteins necessary for assembling the RNA form of the gene transfer vector into infectious
viral particles.
131
Fig. 2. Schematic of EIAV vector system. SD, major splice donor site. pA, polyadenylation signal. R-U5,
sequence domains from EIAV LTR.
possible effects on viral protein expression. Thus the pEV53 viral expression vector contained
an essentially intact 5' untranslated region including a portion of the viral LTR, the tRNA
primer binding site for first strand DNA synthesis and sequences in the 5' leader sequence
predicted for RNA encapsidation.37 Certain cis-acting sequences required for replication were
deleted, however, rendering pEV53 incapable of replication. These include cis-acting sequences
from the 3' end of the genome including the polypurine tract adjacent to the 3LTR used for
initiation of second strand DNA synthesis, and the entire 3' LTR sequence, including cisacting sequences required for integration.
More recent modifications have been made to further disable the replication potential of the
protein expression cassette. This work has also addressed the role of accessory protein expression
for EIAV vector production. Deletion of the 5' leader region, including all LTR sequences and the
tRNA primer binding site up to the major splice donor sequence can be accomplished without
adversely affecting expression of gag-pol in human 293 cells (M Patel and JC Olsen, unpublished
data). This deletion also spans the first exon of tat. Since Tat transactivation of the viral LTR has
been shown to be non-functional in human cells, this finding is not surprising. However, exclusion
of tat from the vector system is desirable since this eliminates any possible unknown adverse affects
that might be caused by delivery of Tat to host cells.
Mitrophanous and colleagues have shown that S2 gene can be eliminated from the vector
system.38 Deletion of S2 sequences from both the protein expression cassette and from the
RRE region of the gene transfer vector had no effect on vector titers or the ability to transfer
genes to growth arrested cells or to cultured rat neurons. This result is consistent with the
results of others who have shown that mutations in S2 have no affect on the ability of the wildtype virus to infect equine cells in culture, including monocyte-derived macrophages.6
Since EIAV and other non-primate lentiviruses encode sequences for a dUTPase activity
not contained in HIV-1, it is of interest to determine the role of this gene in vector production.
As previous studies with the wild-type virus had indicated that the dUTPase activity was important for infection of equine macrophages but not dividing monocytes,23,24,30 it was thought
that the dUTPase activity might be generally important for transduction of non-dividing cells.
To test this, the active site for dUTPase activity was mutated in the pONY3.1 protein expression cassette using an identical mutation that had been shown to adversely affect virus replica-
132
tion in quiescent equine macrophage cultures.30 Interestingly, it was found that the EIAV
dUTPase was not required for gene transfer to aphidicolin arrested cells or to rat neurons in
culture.38 It is unknown, however, if the dUTPase activity will be dispensable for all gene
transfer targets or if it will be important for specific applications.
133
using transduction of bone marrow cells for successful treatment of a FA mouse model.45
Clinical trials, however, using MLV vectors have not yet demonstrated lasting improvement.46
Thus the Yamada study represents an initial effort to determine the utility of using lentiviral
vectors for treatment of this disease. EBV-transformed lymphoblasts derived from FA group C
patients were transduced with vectors containing the normal FA-C cDNA. Phenotypic correction of FA cells, as measured by drug resistance to mitomycin C, was demonstrated for EIAV,
HIV and MLV-derived vectors. Cells transduced by all three vectors showed normalized cell
cycle kinetics and significantly less chromosomal damage after exposure to mitomycin C.44
Differences were noted, however, in gene expression by the various vectors. EIAV vectors expressed about one-half to one-third of the steady state levels of RNA in transduced lymphoblasts, compared to HIV or MLV vectors. Also, the EIAV vector directed the synthesis of an
apparently full length RNA at levels about equivalent to RNA directed from an internal CMV
promoter. This result suggests that the EIAV LTR, even in the absence of EIAV Tat, can actively direct transcription in human cells. Thus in order to prevent transcription from the 5'
LTR in transduced cells, which may interfere with transcription from internal promoters, removal of transcription elements from the U3 region to generate self-inactivating (SIN) vectors
seems advisable.
CAEV-Based Vectors
An initial attempt to generate vectors from caprine arthritis encephalitis virus (CAEV)
resulted in vectors that transferred genes with low transduction efficiencies.54 In this study, two
gene transfer vectors were constructed in which reporter genes were used to replace all of the
viral genes from the start of gag to all but about 180 nucleotides of the env gene (Fig. 3). One
gene transfer vector (pBNL2) contained a neo gene expressed from full length RNA transcripts
and a lacZ gene expressed by splicing using the major CAEV splice donor site in the 5' leader
region and a heterologous splice donor site between neo and lacZ. A second vector (pCSHL)
contained the major splice donor, no splice acceptor and a phleomycin-lacZ fusion gene (Fig.
3). For virus production, the gene transfer vectors were first transfected into goat embryo
fibroblasts and cell lines generated using the selectable markers within the vectors. To rescue
vector sequences into virus, the permissive cell lines were infected with a replication competent
134
Fig. 3. Schematic of CAEV gene transfer vectors. SD, major splice donor site. SA, splice acceptor. SH,
phleomycin selection marker.
strain of CAEV and virus stocks were harvested four days later. Rescue of the gene transfer
vectors was found to be very inefficient, resulting in lacZ titers of 10-187 TU/ml for the pBNL2
vector, and 17-40 TU/ml for the pCSHL vector, whereas the helper virus was produced with
titers ranging from 105 to 7x105 TCID50/ml.54
A detailed analysis of vector RNA in CAEV-infected producer cells showed that for the
pBNL2 vector, both vector-length and spliced RNA could be detected by Northern blot analysis of total cellular RNA; however, only spliced sub-genomic RNA was detected in the cytoplasm.54 Subsequent analysis of virion RNA or RNA in viral vector transduced cells confirmed
that the spliced RNA form was packaged, albeit at low efficiency. Vector-length pCSHL RNA
accumulated in the cytoplasm at higher concentrations than vector length pBNL2 RNA, suggesting that CAEV RNA containing a splice donor but no splice acceptor can be exported from
the nucleus. The greater apparent RNA encapsidation efficiency of vector-length pCSHL relative
to sub-genomic spliced pBNL2 RNA might suggest that CAEV sequences downstream of the
splice donor contribute to RNA encapsidation.54
The overall poor vector production by the CAEV vector system might in part reflect the
absence of an RRE within the vector, resulting in poor accumulation of vector-length CAEV
RNA in the cytoplasm.
135
Fig. 4. Schematic of visna virus vector system. Top, an example of a vector used in two-plasmid vector system.
R-U5, sequence domains from visna virus LTR. SD, major virus splice donor site. PGK pro, the murine
phosphoglycerate kinase promoter. Middle and bottom, examples of visna virus protein expression cassette
and gene transfer vectors used in three-plasmid vector system. MND pro, the myeloid proliferative sarcoma
virus promoter.
Constructs were evaluated in a transient 293-cell expression system for cytoplasmic Gag mRNA,
cell-associated Gag protein levels, and virion-associated Gag protein and virion reverse transcriptase activity. It was found that replacing the visna virus LTR with the immediate early
CMV promoter led to a 4-fold increase in cell-associated Gag expression. Virion production
from 293 cells was also found to be very good as assessed by virion-associated Gag protein,
vector length RNA and reverse transcriptase activity. In fact, reverse transcriptase activity was
as good or exceeded reverse transcriptase activity from parallel cultures transfected with a CMVdriven HIV protein expression cassette vector. To assess gene transfer ability, human 293T
cells, human lymphoid CEM cells or sheep choroid plexus cells (permissive for visna virus
replication) were used as transduction targets for GFP-containing vectors. It was found that
while HIV vectors transduced all three cell targets readily, very little transduction was observed
by the visna vectors.55
Further visna virus constructs were made to use in a three-plasmid vector system.55 These
constructs, similar to the design of other lentiviral three-plasmid systems, separated the viral
genes and the gene tranfer vector onto two plasmids (Fig. 4, middle and bottom). The viral
gene expression constructs (e.g., VH2) were deleted of cis-acting sequences including LTR
sequences and the 3' PPT tract required for replication. Gene transfer vectors contained the
136
CMV promoter fused to the visna TATA box, various amounts of sequences from gag to provide an RNA encapsidation signal, and various amounts of sequences from env to serve as a
source of the RRE. Constructs were identified that yielded good levels of vector-length cytoplasmic RNA and, by comparison to HIV derived vector controls, good encapsidation of vector length RNA into virions (e.g., vector VV2-PG, Fig. 4). However, transduction efficiency was
100-300 fold less than expected compared to HIV vector production carried out in parallel.
The mechanism for the low transduction was investigated.55 Improvements in relative
infectivity were not seen when visna vectors were produced in cells permissive for visna infection, suggesting that producing these vectors in human cells was not necessarily detrimental.
Similar levels of VSV-G were found in visna and HIV virion preparations, as assessed by Western blot analysis, so inefficient transduction was not due to lack of G envelope incorporation.
Using a PCR strategy to detect nearly complete DNA products in transduced cells, it was
determined that visna vector transduced cells contained about 30-fold less viral DNA than
predicted. Furthermore the visna vector DNA was 10-fold less efficiently integrated than HIV
vector DNA. Thus the low transduction efficiency appears to be due to problems occurring at
early steps in the transduction process. Once visna vector DNA has integrated, however, expression levels remained stable for at least several weeks.
Bovine Lentiviruses
The bovine lentiviruses contain two members: bovine immunodeficiency virus (BIV) and
Jembrana disease virus (JDV). Despite its name, BIV has been associated with only a mild
clinical syndrome in infected taurine cattle (Bos taurus).56 In contrast, JDV causes a severe
acute febrile illness in infected Bali cattle (Bos javanicus).57 These cattle are raised mainly in the
Jembrana district on the island of Bali in Indonesia.58 The acute disease associated with JDV
infection has a short incubation time (5-12 days) and a duration of about 7 days. Infected
animals show signs of fever, lymphadenopathy and lymphopenia. The mortality rate of infected animals is significant, about 17%.59 The disease is characterized pathologically as an
intense lymphoproliferative disorder in which proliferating infected lymphoblastoid cells are
found in lymphoid tissues of most organs, particularly in enlarged lymph nodes and spleen.59,60
Cellular infiltrates are also found in the parenchyma of other organs including the lungs, liver,
and kidneys.59 In fatal infections death is attributed to multi-organ failure. In non-fatal infections animals recover about 4-5 weeks post-infection and exhibit no more clinical signs. Recovered animals are resistant to further infection.61
137
Fig. 5. Schematic of JDV vector system. SD, major splice donor site. RRE, presumed rev responsive element
from the 3' region of JDV env.
retained in the gene transfer vectors to ensure efficient encapsidation. The sequence at the gag
start codon was mutated by causing a frameshift at that site.
VSV-G pseudotyped JDV vectors were produced having titers in the range of 0.4-1.2 x
106 G418 colony forming units/ml using 293 cells to titer virus.62 Transduction of GFP was
demonstrated for several cell lines including human 293 and HeLa cells, monkey COS7 cells,
murine B16 cells and primary fetal bovine lung cells. These cell types were transduced with
efficiencies ranging from 28-78%, based upon GFP fluorescence, at a relatively low MOI of 5.
The vectors showed a slight preference for fetal bovine lung cells among the cell types tested.
Transduction of aphidicolin treated HeLa cells was as efficient as untreated cells indicating that
JDV vectors can transduce non-dividing human cells.
Future Prospects
In summary, significant progress has been made in deriving gene transfer vector systems
from EIAV, CAEV, visna and JDV. Due to the unpredictability of deriving simple vector systems from complicated viruses, variability in the success of constructing efficient vector systems has been observed. Relatively efficient vector systems have been made from EIAV and
JDV.37,38,62 It is likely that future work will focus on improving the efficiency and safety of
these systems. In contrast, gene transfer titers of vectors derived from CAEV and visna virus
have been disappointing.54,55 It is likely that future work will address potential reasons for this
in more detail. A recent study has shown that VSV-G pseudotyping of otherwise wild-type
CAEV allowed an efficient single round of infection of human cells.63 It is notable that in these
studies human 293 cells were used for the efficient production of VSV-G pseudotyped virus.
Thus, there does not appear to be any obvious obstacle in developing efficient vector systems
from CAEV.
Preliminary reports have appeared describing VSV-G packaging cell lines capable of stable
production of EIAV vectors.64,65 Stable vector producing cell lines will be important for quality
control analyses of cells and vector preparations and for scaling up vector production for human clinical trials. The eventual use of these vectors in clinical trials will depend upon steady
improvement in gene transfer efficiency and the safety validation of these novel vector systems.
138
Note added in proof: A recent study has described the construction of gene transfer vectors
based on bovine immunodeficiency virus.66
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derived from bovine immunodeficiency virus. J Virol 2001; 75:3371-82.
CHAPTER 8
Abstract
Introduction
Retroviruses are small in the genetic sense, having genomes that are about 10 kb with a
relatively small complement of proteins. With such limited genetic information, they depend
Lentiviral Vector Systems for Gene Transfer, edited by Gary L. Buchschacher, Jr.
2002 Eurekah.com.
142
heavily on their host for essential replication functions, all of which are intimately linked to the
host cell. In its proviral form the retroviral genome mimics that of the cell, and gene expression
involves extensive interactions with the host cell machinery. Retroviruses are unique among
infectious agents in the way they interact with the host cell and in the consequences of this
interaction. Retroviruses regularly integrate their genetic information into the host genome;
they can acquire genes into their genome and they can infect the germ line of the host organism. Integrated proviral DNA is permanent and enables coevolution in both the short and long
term. Retroviruses display a remarkable variety of pathogenic interactions with their host organism. Retroviral diseases may be acute, episodic, or chronic, appearing soon after infection
or after a long asymptomatic period.
The ability of retroviruses to stably integrate their genomes into chromosomes of target
cells provides a strong incentive for the development of retroviral-based vectors for gene therapy.
Over the last two decades, retroviral vectors, derived from oncoretroviruses such as Moloney
murine leukemia virus (MoMLV), have been used widely for gene transfer, both experimentally and in clinical protocols.1,2 In clinical protocols, MoMLV has been associated with inefficient gene transfer. This is due, in part, to the inability of oncoretrovirus-based vectors to infect
nondividing or slowly dividing cells.3,4 Although of limited clinical benefit, studies on MoMLV
vectors have important implications for the development and safety design of current retroviralbased vectors, including lentiviral vectors.
The principal consideration regarding safety is the possibility that administration of the
vector may lead to the emergence of replication-competent retrovirus. Safety advancement in
retroviral vector design includes the development of packaging systems that provide all of the
retroviral proteins in trans to a replication-defective gene transfer vector. The packaging component expresses the viral structural proteins required for assembly of the virus particle and is
stripped of cis-acting sequences necessary for the transfer of the viral genome.5 For a review see
Chapters 1 and 4 and reference.6 These cis-acting sequences are retained within the gene transfer vector where they facilitate its encapsidation, reverse transcription, and integration. This
design does not completely exclude encapsidation of other viral and cellular RNA strands,
including that derived from the viral packaging construct. Copackaging of helper and vector
genomes allows genetic recombination to occur during reverse transcription and thus rejoining
of viral protein-coding sequences and cis-acting sequences of the vector. If recombinants are
formed that contain reproductive functions, it is possible they may replicate and spread to infect
other cells. Thus, the inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of retroviral vectors in human clinical protocols.7-9
Several studies have reported the generation of RCR from retroviral packaging cell lines as
a result of genetic recombination between the vector and helper sequences. The most notable
example comes from a study of retrovirus-mediated gene transfer in rhesus monkeys. After
autologous transplantation of bone marrow stem cells that had been transduced ex vivo with
MoMLV vector, three of ten treated animals developed T-cell lymphomas10 and RCR was
isolated from two of the three animals. In one animal the RCR resulted from recombination
between the vector and packaging sequences introduced into the producer cell line used to
generate the vector.11,12 The genome of a second RCR was identified to have arisen by recombination involving the genome of a vector/helper recombinant with that of an endogenous
murine retrovirus present in the producer cell line.12,13 Although the packaging and vector
components were designed to minimize recombination events, the emergence of RCR in producer cells was common in this early type of packaging cell line.14,15
To improve vector safety, third generation retroviral packaging cell lines were designed by
separating the gag/gag-pol, env and vector functions onto different genetic fragments. The
generation of replication competent retrovirus by the third generation GP+envAM12 packaging cell line has been documented, showing that even packaging cell lines with split viral pro-
143
tein-coding regions can recombine to form RCR.16,17 In this case, the RCR was produced by
recombination events at sites of partial homology between sequences in the vector, the packaging construct and an endogenous retroviral element in the producer cell.18 Vector producing
cells that eliminate homology with endogenous sequences (such as HEK-293) and minimize
the amount of overlapping sequence shared between the genomes of the vector and packaging
construct have not been found to generate RCR.19 While the generation of RCR has been
reduced markedly by improvements in vector design and vector producer cells, these results
emphasize the potential for retroviral-mediated recombination.
Because retroviral vectors can be permanently integrated in the host cells chromosomes
and passed to daughter cells, integration itself raises safety concerns at two different levels:
insertional mutagenesis and germ line transduction. All viruses and vectors that randomly
insert their genetic information into the genome of target cells can cause insertional mutagenesis. Insertion of proviral DNA can disrupt normal gene function, cause the inactivation of
tumor suppressor genes or the activation of oncogenes. For reviews see references.6,20 This risk
is directly proportional to the number of insertion/integration events. Although the activation
of a protooncogene by retroviral insertion is not itself usually sufficient to convert a normal cell
into a tumor cell it can be a rate-limiting step. In the case of oncoretroviruses, insertional
mutagenesis may be largely due to relatively high numbers of infected cell/insertion events.
Thus, the probability of insertion near a protooncogene is relatively high. By analogy, if genetic
recombination was to produce virus that could replicate and spread, the likelihood of inducing
insertional mutagenesis would increase. The analysis of various tissues from a rhesus monkey
that developed T-cell lymphoma from RCR revealed a common recombinant proviral insertion
site, suggesting that chronic productive retroviral infection leads to insertional mutagenesis of
critical growth control genes.12 There is no evidence to date that primary vector-transduced
cells have had adverse effects in animals or humans. Provirus formation in a germ cell provides
the means for retroviruses to colonize the germ line of the host. Extensive analysis in animal
models and human trials has not demonstrated transmission of retroviral vectors into the germ line.
Based on studies with retroviral vectors it is clear that RCR can be generated from retroviral
packaging cell lines through genetic recombination between the gene transfer vector and transpackaging genetic elements. Moreover, endogenous retroviral sequences may contribute to the
emergence of RCR. Therefore, our primary consideration for safety, and the principal focus of
discussion in this Chapter, is a lentiviral vector design as an example of an approach that might
minimize the risk that genetic recombination may lead to the emergence of RCR. Discussion
of other safety considerations involving the use of lentiviral vectors may be found in Chapters
4 (HIV-1 Vectors), 9 (Prospects for Gene Therapy) and 10 (Ethical Considerations).
144
Fig. 1. Overview of method for monitoring the generation of env-minus recombinant lentivirus. (A)
Through nonspecific encapsidation, other RNA molecules, including those derived from the packaging
construct, may be co-packaged with the gene transfer vector RNA. Preparations of vector stocks are used
to transduce cultures of 293T cells (MOI=2). (B) Genetic recombination between RNAs of the packaging
construct and the vector may generate env-minus recombinant lentiviral genomes. If stably integrated into
the chromosomes of transduced 293T cells, such recombinant proviral genomes may express viral proteins
and produce env-minus progeny lentiviral particles that package the recombinants RNA genome. To detect
these recombinants, the transduced 293T cells are cotransfected with a VSV-G expression plasmid to
pseudotype progeny virions and a tat expression plasmid to up-regulate expression of recombinant proviral
genomes. (C) Three days later, the culture supernatants are concentrated by ultracentrifugation. Progeny
virions with encapsidated recombinant genomes are depicted. (D) The pseudotyped virions are then used
to infect HeLa-puro cells and 30 hours later the cell monolayers are trypsinized and replated in medium
containing puromycin. Pseudotyped particles confer resistance to puromycin if they contain a recombinant
lentiviral genome that is reverse transcribed, integrated, and expresses Tat protein.
145
limitations of animal models to evaluate lentiviral vectors for their potential to induce disease,
the vector design itself should ensure the greatest level of safety that is achievable.
Principally, lentiviral vector systems are comprised of three separate parts: the packaging,
gene transfer vector, and envelope (env) components. The most advanced third generation
packaging construct includes many safety design features. For the HIV-1 based vector, all of
the accessory genes (vif, vpr, vpu, nef) have been deleted. In addition, the two key regulatory
genes, tat and rev, have been either deleted or separated from the packaging construct.30-33 See
Chapter 4 for a review of HIV-1 based vectors. With only the gag and pol genes remaining, it is
not possible for genetic recombination to generate a virus with pathogenic features like that of
the parental virus. Importantly, the third generation packaging construct has not diminished
vector titer. Safeguards have also been built into the gene transfer vector. The vector contains all
of the cis-acting elements necessary for packaging, reverse transcription, and integration, while
all viral open reading frames have been eliminated. The self-inactivating design (SIN) feature
deletes the viral transcriptional promoter and enhancer elements. Self-inactivation relies on a
deletion made in the U3 region of the 3' LTR of the DNA that produces the vector RNA. By
reverse transcription, the 3' U3 deletion is duplicated in the 5' LTR. Consequently, transcription
of full-length vector RNA is markedly reduced in cells transduced with a Sin vector.34,35 This
further minimizes the risk for generating RCR and reduces the chance that cellular coding
sequences located adjacent to the integrated vector will be aberrantly expressed due to promoter activity of the 3' LTR. With the deletion of the viral transcriptional elements from the
vector, synthesis of vector RNA will largely depend on the site of vector integration and surrounding cellular transcription elements.34
Several in vitro assays have been used to evaluate the safety of HIV-based vectors, including marker rescue, tat-transfer and gag-transfer assays. These assays have not provided any
evidence of either RCR or genetic recombination.28,29 To fully assess the safety risks, we thought
it was necessary to understand whether recombination occurs between the genetic components
of the lentiviral vector system, and if so, to characterize the nature of the recombinants at the
molecular level. Then it would be feasible to design and evaluate new approaches that overcome the safety risks associated with genetic recombination. A highly sensitive biological assay
was developed to specifically detect vector recombination in transduced cells, independently of
the generation of RCR.36 This assay is based on a cell line containing a stably integrated copy
of the puromycin resistance gene introduced by transduction with a lentiviral vector. Since the
puromycin gene was placed under control of the HIV-1 LTR, it is inducible by Tat expression.
An overview of this assay is depicted in Figure 1. Supernatants from cultures of 293T cells
infected with a second generation lentiviral vector (107 infectious units [IU]) and then
cotransfected with VSV-G (pMD.G) and tat (ptat) expression plasmids were analyzed for recombinant virus by incubation with the puromycin inducible cell line (Fig. 1C). The detection
of puromycin resistant cell colonies suggested the formation of vector/helper recombinants
(Fig. 2). The non-nucleoside reverse transcriptase inhibitor, Nevirapine, completely blocked
colony formation, indicating that the formation of lentiviral vector recombinants was dependent on the function of the HIV-1 reverse transcriptase. The generation of resistant colonies
was also completely dependent on pseudotyping with the VSV-G protein, demonstrating the
recombinant virus was defective in env. Transfection of the VSV-G expression plasmid for
pseudotyping virions generated from recombinant lentivirus is unique to this assay approach
and importantly, it allows for the monitoring of env-minus recombinant lentivirus instead of RCR.
The formation of recombinant provirus was confirmed by genetic sequence analysis.36
The 5 sequence consisted of U3, R, U5, and the packaging signal (derived from the vector)
joined with the gag open reading frame (derived from the packaging construct). Analysis of 3
sequences also revealed a physical linkage between the packaging construct and the gene transfer vector. Interestingly, the point of vector recombination occurred within the poly(A) tract of
146
Fig. 2. Detection of env-minus recombinant lentivirus. Supernatants from cultures of 293T cells transduced with 107 infectious units (IU) of the lentiviral vector were used to infect cultures of HeLa-puro
cells. One set was infected two hours after adding Nevirapine (5 uM) to the culture medium. The control
culture contained uninfected HeLa-puro cells. After selection in medium containing puromycin, the cell
monolayers were visualized by staining with crystal violet. The formation of resistant colonies was
documented by microscopy.
the packaging constructs mRNA (Fig. 3). This finding confirmed that the env-minus recombinant lentivirus was generated during reverse transcription in primary transduced cells. Such
non-homologous genetic recombination suggests that a similar mechanism could occur in the
more advanced third generation vectors. This assertion is strengthened by earlier studies that
demonstrated avian retroviruses capture oncogenes through non-homologous recombination
within the poly(A) tract.37,38 Using a modified version (Tat-independent) of the above DNA
mobilization assay, preparations of third generation and SIN vectors were analyzed and found
to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when
pseudotyped with an exogenous envelope protein (Fig. 4). Taken together, these data indicate
the generation of env-minus recombinant provirus, expression of the recombinants genes, assembly of lentivirus-like particles, and encapsidation and mobilization of viral nucleic acids to
new host cells when an exogenous Env is provided in trans. These findings underscore the
significance for understanding lentiviral vector genetic recombination and reemphasize the
importance for thorough examination of possible safety risks while advancing lentiviral vector
toward human trials.
A safety design was recently introduced that may largely overcome the risks that stem
from genetic recombination. Based on the ability to rescue the infectivity of RT-IN deleted
HIV-1 by providing the RT and IN proteins in trans,39-42 a new class of HIV-based vectors was
designed that splits the gag-pol component of the packaging construct into two separate parts:
one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr
(Vpr-RT-IN) (Fig. 5). Removing RT and IN from the other components of the packaging
construct disarms the retroviral replication machinery conserved within the gag-pol structure.
This vector (termed trans-vector) was evaluated in side-by-side experiments with third generation and SIN vectors for its ability to recombine and mobilize DNA. While the third generation and SIN vectors transferred puromycin resistance to naive cells, the trans-vector did not
(Fig. 5). This suggests that the trans-vector design prevents the generation of recombinant
147
Fig. 3. Genetic analysis of env-minus recombinant lentivirus. High molecular weight DNA prepared from
puromycin resistant cells was subjected to PCR using primer pairs specific for sequences of the vector and
packaging construct. (A) Sequence analysis of the 5 end of the recombinant genome. Using a sense primer
specific for the U3 region of the 5 LTR of the gene transfer vector and an antisense primer specific for 3
gag sequences of the packaging construct, a PCR fragment of approximately 2000 base pairs was amplified,
cloned and sequenced. The nucleotide sequence at the junction of the vector and gag gene is illustrated. Of
10 clones analyzed, the sequence was identical. (B) Sequence analysis of the 3 end of the recombinant
genome. Using a sense primer specific for the first tat exon of the packaging construct and an antisense
primer specific for the 3 U3 region of the vector, a DNA fragment of approximately 2500 bp was derived
for sequencing. In all of the clones that were analyzed, the 3 end of the vector was found to be joined with
the poly(A) tract of the packaging construct. The nucleotide sequence between the U3 region of the vector
and the poly(A) tract of the packaging construct is illustrated. The genotype of four clones among nine that
were analyzed is depicted. (C) Genetic recombination during reverse transcription. The diagram illustrates
how the recombinants (depicted in B) were likely generated during synthesis of the negative-strand DNA.
148
Fig. 4. Analysis of state-of-the-art lentiviral vector systems for the generation of env-minus recombinant
lentivirus. Stocks of lentiviral vectors were prepared using a third generation packaging construct (A), a third
generation packaging construct in combination with a Sin vector (B), and the trans-vector (C), respectively.
108 infectious units of A and B and 109 infectious units of C were used to infect cultures of 293T cells
containing the puromycin resistance gene as depicted in Figure 1. Three days later the culture supernatants
were collected, concentrated by ultracentrifugation and used to infect HeLa-tat cells. After selection in
medium containing puromycin the resistant cell colonies were visualized by staining with crystal violet.
149
Fig. 5. Genetic components of the trans-lentiviral packaging system. The trans-lenti packaging construct
is illustrated as pCMV-gag-pro. The pCMV-vpr-RT-IN construct encodes the Vpr-RT-IN fusion protein, which is packaged into the Gag/Gag-Pro particles, providing the reverse transcriptase and integrase
function. Proteolytic processing by the viral protease liberates mature and enzymatically active RT (p51/
p66) and IN proteins.41
Quality assurance methods based on in vitro monitoring for RCR seem to be of limited
value as a predictor of safety in vivo, especially in the long term. The detection of env-minus
recombinant lentivirus among 107 infectious vector particles raises the possibility that in the
large scale production required for gene therapy applications, the probability of generating
recombinants containing functional env may be increased. The ability of HIV-1 cores to acquire
and utilize cellular membrane proteins such as CD4/CCR5 or CXC4 for infection47-49 suggests
the possibility that env-minus recombinant virus could infect dividing and nondividing cells
through alternative mechanisms (Env independent). It has long since been thought that infection
with xenotropic endogenous retroviruses may have occurred via alternative viral or cellular
receptors provided in trans.50 This notion was recently supported by a report showing envminus HIV-1 can infect CD4-minus and CD4-positive cells through a pathway independent
of the viral Env glycoprotein.51 Therefore, the ability to directly monitor env-minus recombinant
virus in vitro as a surrogate marker for the possible emergence of RCR in vivo may represent a
significant advancement in quality assurance. The combination of the trans-vector design with
an in vitro assay that monitors for recombinants devoid of functional gag-pol as a means to
quality assure lentiviral vector stocks is illustrated in Figure 6.
The failure to detect env-minus recombinant lentivirus in the trans-vector system may
suggest that either the assay is not sensitive enough or that recombinants are not formed at the
titers tested. By comparison with lenti-vectors, the assay is at least three orders of magnitude
beyond the limits of sensitivity. Since the trans-vector has been show to generate RT-IN minus
recombinants,36 it may also be possible to regenerate recombinants containing functional gagpol. However, this would require three restricted steps: first, single virions (vector particles)
must co-package three different mRNAs, two of which do not contain the packaging signal;
second, the three separate genetic elements (mRNAs) must recombine; and third, recombination
must occur in a manner that restores a functional LTR-gag/gag-pol-LTR structure. The failure
to detect recombinants with functional gag-pol in large-scale, high titer stocks (>109) of transvector support the idea that the trans-lenti design help control the risks associated with genetic
recombination.
150
Fig. 6. Monitoring for env-minus recombinant lentivirus. The diagram illustrates how the in vitro analysis
of vector stocks for env-minus recombinant lentivirus may serve as a surrogate marker for the emergence
of RCR in vivo. In the absence of detecting recombinants (LTR-gag-pol-LTR) there would be a greatly
diminished chance of generating RCR.
Previous studies indicated that endogenous retroviral sequences may recombine with
retroviral vectors and lead to the generation of RCR. This raises the issue of whether RT and
IN derived from endogenous retroviruses could complement RT-IN minus trans-vector recombinants. Recently, the protease of the human endogenous retrovirus HERV-K was analyzed for its ability to process the HIV-1 Gag and Gag-Pol precursor polyproteins by targeting
the HERV-K protease into immature HIV-1 virions. This analysis demonstrated that the HERVK protease was severely defective in the proteolytic processing of the Gag and Gag-Pol precursors.52 Even the closely related HIV-2 RT protein is not correctly processed by the HIV-1 PR
(unpublished). These results suggest it is unlikely that endogenous RT and IN could rescue
RT-IN defective recombinants (LTR-gag-pro-LTR).
Non-human primate lentiviruses, including equine infectious anemia virus (EIAV), feline
immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV) are being considered as gene therapy vectors,26,53,54 primarily because they are not associated with disease in
man. Significant improvements have recently been made in these vector systems (see Chapters
6 and 7). By analogy with the HIV-1-base lentiviral vector, it is unlikely the current design of
non-human primate vectors will prevent the generation of recombinant virus. Since the design
of the trans-vector packaging construct (Gag-Pro) is based on supplying RT and IN in trans as
fusion partners of Vpr/Vpx and similar virion-associated accessory proteins are not encoded by
the non-human lentiviruses, the trans-vector design would not be directly applicable to other
lenti-vectors. However, the development of alternative approaches to disarm the gag-pol structure
of these viruses would seem feasible.
151
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CHAPTER 9
Abstract
ecombinant vectors derived from murine leukemia virus (MLV) have been widely used
to introduce genes in human gene therapy clinical trials and have shown the potential
for medical applications and the promise of significantly improving medical therapies.
Yet, the demonstrated limitations of these vectors support the need for continued development
of improved vectors. The intrinsic properties associated with the MLV genome and its life cycle
do not favor the successful application of this vector system in certain human gene transfer
applications. Since MLV integrates randomly into the host genome, transgene expression is
frequently affected by the flanking host chromatin.1 MLV insertions can often result in silencing or position effect variation of gene expression either immediately after insertion or following cell expansion in culture or in vivo.2-6 Migration of the MLV pre-integration complex from
the cytoplasm into the nucleus of infected cells requires mitosis for nuclear membrane breakdown.7 Since a majority of human cells exist in a quiescent state in vivo, it is unlikely that direct
in vivo gene delivery into target tissues can be achieved with the MLV vector system. Finally,
insertion of tissue-specific cis-regulatory sequences to direct transgene expression frequently
results in either the rearrangement of the vector sequence or disruption of the cis-regulatory
sequence functions.8-12 The long terminal repeat (LTR) of MLV, which contains a ubiquitously
active enhancer/promoter element, may partially account for this problem. Together, these
problems pose a major obstacle for the use of MLV vectors in the treatment of human diseases.
This Chapter discusses some of the potential targets to which HIV vectors might be applied in
clinical settings and some of the issues surrounding use of HIV vectors in gene transfer clinical trials.
154
tional advantage of using HIV vectors is the ability to accommodate inserted cis-regulatory
sequences to direct transgene expression.15,29,30 This is in contrast to the observation that insertion of similar sequences into MLV vectors frequently leads to vector genome rearrangement
and functional loss of the cis-regulatory sequences.8-12 Although the reasons for this difference
between the two vector systems remain unclear, it is likely that sequence elements present in a
MLV vector are not always compatible with an inserted cis-regulatory sequence. While several
major obstacles exist that may impede the start of human gene transfer clinical trials, such as
isolation of stable packaging cell lines for efficient scale-up of vector production and establishment of processes for bulk vector purification and of standardized and sensitive procedures for
the detection of replication competent retrovirus (RCR), the advantages of HIV vectors open
exciting perspectives for using this vector system to ultimately treat human diseases.
155
protein-replete counterpart. These observations, however, do not exclude the possibility that
the accessory proteins may contribute in subtle ways to enhance the transduction efficiency of
specific cell types. Kafri et al reported that the use of a Vpr(-)/Vif(-) HIV vector in vivo resulted
in a significant decrease in its ability to transduce liver cells.14 Gasmi et al also observed that the
complete absence of all four accessory proteins resulted in a decrease in the ability of an HIV
vector to transduce quiescent primary human skin fibroblasts.33 Chinnasamy et al demonstrated that HIV vectors without the accessory proteins failed to integrate in resting lymphocytes.40 Thus, the accessory proteins may facilitate efficient transduction of HIV vectors under
certain conditions. Nevertheless, complete elimination of accessory proteins from HIV vector
preparations seems to have little effect on titers and should significantly enhance the biosafety
of this vector system in human clinical trials.
156
homology with any naturally occurring HIV-1 gag-pol sequence, the possibility of RCR production through homologous recombination between the packaging plasmid and the vector is
also minimized. While vector titers generated from Rev-independent packaging plasmid were
somewhat lower than that from Rev-dependent packaging plasmid, this study clearly demonstrates the possibility of generating HIV vectors with only two of HIV-encoded proteins: Gag
and Pol.46 Such a Rev-independent Gag-Pol expression system, coupled with the observation
that multiple vector sequence modifications eliminated the requirement of RRE and Rev for
the production of the vector genomic RNA,53 should significantly alleviate the concern of
applying HIV-based vectors in the treatment of human diseases.
The presence of only the gag and pol genes in the HIV vector production system makes it
even less likely to produce RCR than the MLV vector production system since HIV replication
is critically dependent on Tat and Rev. Complete removal of the HIV promoter and enhancer
sequences in SIN vectors further reduces the likelihood of RCR production, either through
recombination between the packaging plasmid and the vector construct or through fortuitous
co-packaging of RNAs derived from the packaging construct and the vector construct into the
same virion. In this regard, the MLV vector system may be more likely to generate RCR, since
the MLV promoter and enhancer sequences do not depend on any viral-encoded proteins for
function and the LTR is transcriptionally active in most cell types. Since only Gag, Pol and
VSV-G are required for the packaging of the HIV vector genomic RNA, stable packaging cell
lines might be established. The problem of cell toxicity caused by stable expression of Vpr and
Nef is therefore avoided.54-59 The presence of the stable packaging cell lines might not only
facilitate large-scale vector production, but also minimizes the possibility of RCR production
through DNA recombination during transient transfection for vector production. While VSVG expression is toxic to mammalian cells, inducible expression of this protein in established
stable cell lines has been demonstrated before.60,61 With the experience in MLV vector production, it is very likely that stable HIV packaging cell lines that generate reasonably high vector
titers can ultimately be established and used in human gene therapy clinical trials.62-64
RCR Detection
Given the limitations of available animal models for HIV infection, the biosafety of HIV
vectors will ultimately be determined in human patients. Thus, sensitive assays need to be
established to detect the presence of RCR in vector preparations. The HIV p24 ELISA assay to
detect the viral capsid protein is an extremely sensitive assay. The amount of p24 as low as 10
picogram can readily be detected with this assay system. To increase the sensitivity, a certain
portion of the vector preparation is first allowed to infect a permissive cell line for HIV replication, such as human T cell lines. The infected cells are serially passaged to allow sufficient time
for the amplification of contaminating RCR that may be present in very low abundance. The
culture supernatant is harvested and virions in the culture supernatant are concentrated by
ultracentrifugation and detected by the p24 assay kit.
An alternative approach to detect RCR is using the marker rescue assay. A human cell line
containing an integrated HIV vector with a selectable marker gene such as that encoding
neomycin phosphotransferase (neo) can first be established. This cell line is transduced at high
multiplicity of infection (MOI) with the vector preparation containing the gene of interest. If
RCR is present in the vector preparation, the Neo vector would be rescued and released into
the culture medium. The presence of the rescued vector can be detected by transduction and
selection in G418-containing medium for colony formation. The sensitivity of such an assay to
detect RCR, however, remains to be established. Ultimately, both the p24 assay and the marker
rescue assay may be combined together to increase the sensitivity of detecting RCR in vector
preparations. Further discussion regarding detection of RCR can be found in Chapter 8.
157
158
Retinitis pigmentosa (RP), one of the most common forms of retinal dysfunction, is a
result of photoreceptor cell degeneration. While various genes presumably are involved in the
development of RP, mutations in the rod photoreceptor cGMP phosphodiesterase subunit
(PDE) gene are found in patients with autosomal recessive RP as well as in rd mice and rcd1
Irish setters.72-75 Miyoshi et al injected HIV vectors into the subretinal space of rat eyes and
demonstrated efficient transduction of both photoreceptor cells and retinal pigment epithelium.15 Based on this result, Takahashi et al evaluated RP progression in rd mice by the delivery
of the PDE cDNA via HIV vectors.76 Several rows of photoreceptor cells were detected in
PDE vector-treated rd mice for at least 24 weeks post-injection, whereas no photoreceptor
cells remained in the eyes of control animals 6 weeks post-injection. PDE expression was also
detectable in the photoreceptor cells of PDE vector-treated rd mice. Thus, besides treating
CNS diseases, HIV vector-mediated gene delivery may also be employed to treat inherited
retinal degeneration.
159
160
115
HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. Cells non-productively infected with HIV-1 have been
identified following infection in vitro and in vivo. These cells may shelter the virus from antiviral therapy.87,116,117
The problem of applying these genetic strategies using HIV vectors is that there is the
possibility that the anti-HIV gene will interfere with the production of the vector. For example,
if the gene targeted by the anti-HIV strategy is required for genomic RNA production and/or
packaging, then there will be decreased vector titer. Without efficient vector titers, transduction of hematopoietic progenitor cells or T lymphocytes would be limited. It is even possible
that targeting integrase with SFv will adversely effect not only vector production but also subsequent cellular integration. Therefore, targets such as Rev, RRE, or Gag-Pol would be problematic, but agents that target Tat, any accessory protein, or envelope should not interfere with
vector production or subsequent transduction.
Regulatory Issues
General Considerations
The unrealistic expectations associated with gene transfer research in the early 1990's
generated concern that this area of research needed to be assessed more realistically. Because of
the perception that unrestrained public pronouncements regarding gene therapy studies had
unrealistically influenced the field, there was concern about the overall quality of this research.
An expert panel reviewed the field and made several recommendations [see http://www.nih.gov/
news/panelrep.html]. The panel suggested that this research be considered human gene transfer research since the terminology gene therapy suggested potential effects on disease that
might be unlikely to occur at this stage of development. In addition, it was recommended that
there be a better focus on the basic science aspects of this research, including more pathogenetic studies and better use of animal models. Furthermore, the panel suggested the need for an
improvement in the quality of gene therapy protocols and in the training of those performing
the clinical studies.
In 1999, after there had been hundreds of human research subjects safely enrolled into
human gene transfer research protocols, there were two events which changed the way this
research is regulated. The first was the death of a young man of liver failure after being treated
with a direct infusion of an adenovirus encoding an enzyme involved in ornithine metabolism.
The second was a report of a protocol violation regarding the treatment of a patient with an
adenovirus encoding a potential angiogenesis enhancement factor at a time when the subject
had a lung cancer which was an exclusionary criterion. Investigation of these incidences led to
the conclusion that correct oversight of these important clinical protocols might not have been
adequate. Furthermore, because both studies were conducted at prominent medical centers,
the reviews raised the question of whether the conduct of gene transfer research was adequate
in the multiple other centers conducting such studies. The Food and Drug Administration
(FDA) randomly inspected approximately 10% of all gene transfer studies and found no significant other problems. Nevertheless, the potential for serious malfeasance was established,
and therefore in early 2000, new rules were developed at both the NIH and at FDA for human
gene transfer research. Heretofore, phase I studies had not been as rigorously held to good
clinical practices as phase III trials, but beginning in 2000, both the FDA and the NIH inaugurated changes for improved research subject protection.
161
Federal Review
Review of human gene transfer clinical protocols involves both federal and local review,
but for institutions receiving NIH support, the review must begin with the submission of a
proposal to OBA. OBA is responsible for monitoring all scientific progress in human genetics
research and for facilitating a public discussion regarding the ethical, legal, and social concerns
for research involving recombinant DNA, genetic testing, and xenotransplantation. In this
regard, OBA manages the operation of the RAC that in turn monitors scientific progress in
basic and clinical research involving all recombinant DNA and human gene transfer. Recommendations of the RAC are required before the local review committees can approve any gene
transfer protocol involving human subjects.
The review by the Food and Drug Administration occurs with the Investigational New
Drug (IND) application and with pre-IND meetings with the sponsor of the research. The
IND is a request for waiver from laws that require that only approved drugs be used in treating
patients. The IND provides a complete description of the source and production of the drug
and the approved protocol under which it will be used. The description of specific recommendations for IND preparation can be obtained from the FDA website (http://www.fda.gov/
cber/guidelines.htm)
162
E and K of the NIH Guidelines. Thus, this committee provides a means for verifying that the
federally mandated rules are being implemented correctly at the local level.
The IRB is responsible for review of human subject research to assure that it is scientifically sound, ethical, and safe. The IRB mandate is defined in the code of federal regulation
(CFR), e.g., see 45 CFR 46. In addition, many institutions have a scientific review committee
that provides guidance on adequacy of the study design.
Acknowledgements
The authors are grateful for the technical assistance of Ms. Irene Tomeck in the preparation of this manuscript. This work was supported in part by USPHS Grants No. 5P01 A146030,
and P01 30206-20, and by grant M01 RR-43 from the GCRC Branch of the National Center
for Research Resources, NIH.
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186
Index
A
Accessory proteins 12, 20, 28, 49, 56, 58, 95,
96, 104, 106, 108, 109, 111, 112, 150,
154, 155, 162
AIDS 13, 29, 59, 65, 69, 79, 80, 81, 82, 83,
95, 104, 154, 159
Assembly 5, 22-26, 28, 38, 43, 101, 142, 146,
148
C
CAEV 98, 102, 126, 128, 129, 133-135, 137
Caprine arthritis encephalitis virus 126, 133
CD4 13-17, 27-29, 56, 81-83, 86, 88, 96, 97,
126, 149, 154, 159, 179
Central termination sequence 54
Cis-acting sequences 9, 10, 53, 71, 87-89,
105, 130, 131, 135, 141, 142
constitutive transport element 22, 60, 90
Coreceptor 12, 15-17, 29, 81, 82, 92
CPPT 34, 41, 43, 44, 50, 53, 54, 65
CTE 22, 60-63, 65, 66, 90, 105-107,
110-112
CTS 19, 50, 53, 54, 65
Cytoskeleton 34, 41, 43
D
DUTPase 128, 129, 131, 132
E
EIAV 38, 98, 101, 102, 126, 128-133, 137,
150
Encapsidation signal 53, 90, 130, 136
Env 2, 3, 5-9, 12-14, 16, 17, 21, 22, 25, 27,
28, 49-51, 55-61, 63, 66, 69, 84, 85, 88,
89, 98, 102, 103, 111, 112, 115, 126,
128, 130, 132-134, 136, 141, 142,
145-147, 149, 155
Equine infectious anemia virus 28, 98, 126,
150
F
Fanconi anemia 132
G
G-to-A mutations 129
Gag 5, 7, 12, 19, 21, 22, 24-29, 49, 50,
52-54, 56, 58, 61, 63, 65, 67-69, 80, 86,
90, 100, 101, 103, 106, 110, 112, 130,
135, 141, 146, 148, 150, 155, 156
Gag-Pol 5, 49, 50, 52, 61, 63, 67, 130, 150,
156
Gag-Pro 63, 141, 146, 148, 150
Gene therapy 10, 12, 25, 57, 68, 79, 80, 88,
91, 95, 104, 108, 113, 114, 132, 142,
143, 149, 150, 153, 156, 160, 169, 170,
176, 177, 178, 180, 181, 184
Gene transfer vector 50, 51, 53, 55, 56,
58-66, 68, 130, 131, 133, 137, 142, 143,
145, 146
H
Helper cells 8
Helper virus 8, 88, 134, 162
Hematopoietic stem cells 55, 148
HIV 3, 12-29, 33, 34, 36-38, 40, 41, 43, 44,
48-61, 63-69, 79-91, 95-98, 100-107,
109, 110, 112, 126, 128, 131-133, 135,
136, 141, 143, 145, 146, 149, 150,
153-60, 162, 178, 179, 181, 182
HIV-1 3, 12-15, 17, 19-29, 33, 38, 40, 43,
44, 48-61, 63-69, 79-87, 90, 91, 96, 97,
100, 101, 103, 104, 109, 110, 126, 128,
131, 143, 145, 146, 149, 150, 153, 155,
156, 159, 160, 178
HIV-2 24, 26, 27, 29, 40, 63, 79-89, 91, 126,
150
I
Integrase 3, 4, 12, 33, 34, 36, 37, 40, 43, 48,
50, 54, 67, 80, 83, 100, 101, 114, 148,
153, 159, 160
Integration 4-6, 9, 10, 17, 19, 20, 33, 34,
36-38, 40, 41, 43, 44, 48, 50, 53, 54, 59,
63, 64, 67, 79, 80, 87-89, 104, 105, 130,
131, 142, 143, 145, 153, 155, 160, 178
187
Index
PIC 17, 19, 20, 22, 27, 28, 34, 36-38, 40, 43,
44, 48, 50, 53, 54, 56, 79, 80, 86
Pol 5, 7, 12, 21, 22, 26, 49, 50, 52, 61, 63,
67, 68, 100, 130, 141, 146, 150, 155,
156
Pr160Gag-Pol 49, 50
Pr55Gag 12, 22, 49
Preintegration complex 4, 17, 37, 43, 50, 143
Production 4-7, 9, 10, 22, 24, 41, 52, 55-64,
66, 68, 69, 82, 87, 89-91, 104, 105,
108-112, 114, 129-131, 133-137, 141,
142, 148, 149, 152, 154-156, 160-162,
172, 178, 179, 183
Protease 3, 5, 12, 25, 50, 63, 67, 98, 100,
101, 148, 150, 159
Provirus 2-5, 8, 19, 20, 40, 49, 60, 63, 65,
143, 145, 146, 178
Pseudotyping 9, 25, 55, 56, 59, 88, 106, 107,
132, 137, 141, 145
RCR 62, 68
Recombination 7, 9, 19, 59, 60, 63, 68, 81,
87, 89, 90, 104, 105, 110, 112, 130, 141,
142, 143, 145, 146, 149, 154, 156, 162,
178
Retroviral genes 98
Retroviral vectors 6-9, 79, 88, 91, 104, 106,
109, 142, 143, 150
Retrovirus classification 2
Retrovirus replication cycle 2, 4
Rev 12, 20-22, 27, 29, 44, 49, 50, 52-56,
58-63, 65, 66, 69, 84-86, 88, 90, 98,
101-103, 105-107, 109-112, 128, 130,
134, 136, 145, 155, 156, 159, 160
Rev-independent 60-62, 65, 66, 105, 155,
156
Reverse transcription 2-4, 6, 7, 10, 17, 19, 33,
34, 36-38, 41, 43, 44, 48, 50, 53, 56, 64,
68, 87-90, 98, 129, 141, 142, 145, 146,
154, 155
RNA transport 61, 63
RT 3, 5, 12, 17, 19, 27, 36, 37, 50, 56, 63,
98, 100, 101, 128, 141, 146, 148, 149,
150, 159, 162
N
Nef 12, 13, 21, 27-29, 49, 56-58, 69, 84-86,
88, 89, 128, 145, 154, 156
Neurons 38, 66, 89, 90, 97, 109, 131, 132,
157
NLS 34, 35, 37, 38, 40, 41, 43, 86, 153
Non-primate lentiviruses 40, 41, 80, 98, 100,
126, 128, 131
Nuclear localization signal 20, 22, 91
Nuclear transport 4, 34, 43, 86
O
Oncoretrovirus 1, 142
P
Packaging cells 8, 9
Packaging construct 52, 63, 87, 88, 90, 110,
111, 136, 141-143, 145, 146, 148-150,
156
Packaging signal 5, 12, 23, 24, 53, 88, 104,
105, 106, 111, 112, 141, 145, 149
Pathology 81, 82
S
S2 gene 131
188
Safety 7, 9, 60, 63, 65, 68, 69, 89, 90, 91, 95,
104, 105, 106, 107, 110, 112, 137,
141-143, 145, 146, 148, 149, 154, 161,
162, 177
Self-inactivating (SIN) vectors 7, 54, 63, 90,
133, 155
self-inactivating vector 67
SIN 7, 54, 63, 64, 90, 91, 105, 133, 141,
145, 146, 149, 155, 156
Sin vector 7, 64, 90, 91, 141, 145, 149, 155
T
Tat 12, 20, 21, 27, 48-50, 53-56, 58-61, 63,
64, 66, 68, 69, 84-88, 102, 128, 130,
131, 133, 136, 145, 146, 149, 155, 156,
159, 160, 162
Tat-independent 56, 59, 61, 146
Trans-vector 141, 146, 148-150
Transgene 50, 51, 53, 54, 56-59, 64-69, 91,
105, 109, 110, 113, 130, 153, 154, 155,
157, 161
Transport elements 60, 62
V
Vector 1, 2, 6-10, 12, 29, 33, 43, 48, 50, 51,
53-69, 79, 80, 83, 86-91, 95, 104-114,
126, 129, 130-137, 141-143, 145-150,
153-158, 160-162, 169, 170, 174,
177-179, 181, 182
Vif 13, 21, 28, 29, 49, 50, 56, 58, 59, 96,
102, 103, 106, 109, 110, 128, 154, 155
Visna virus 126, 128, 129, 133-135, 137
Vpr 13, 17, 19, 21, 27-29, 33, 34, 36, 37, 40,
41, 43, 48-50, 56, 58, 59, 63, 67, 80, 86,
109, 141, 146, 148, 150, 153-156
vpr 38, 69, 84, 86, 88-90, 145, 148
Vpr-RT-IN 63, 146, 148
Vpu 13, 21, 27, 28, 49, 50, 56, 58, 59, 69,
109, 154
Vpx 63, 80, 86, 150
vpx 84, 86, 88, 89
W
Woodchuck Post-Transcriptional Regulatory
Element 65
WPRE 65, 105