Final Exam

Monday, December 18, 2:00-5:00 pm

BUR 108
Office hours:
12/13 W 2-4 pm,
12/15 F 1-3 pm
 Molecular Biology of HIV
•  www.hopkins-aids.edu/hiv_lifecycle/
•  www.who.int/hiv/en
•  hivinsite.ucsf.edu/InSite.jsp?doc=kb-02-01-02

HIV
budding
from an
infected
cell.
 A Brief History of AIDS
1981
CDC reports unusual incidences of pneumonia caused by Pneumocystis
carinii and of skin cancers, Kaposi's sarcoma. Patient's immune system
were impaired.

1982
CDC recognized a new disease: Acquired Immune Deficiency Syndrome
(AIDS). Don Francis suggests screening blood for hepatitis C as a
surrogate test for the AIDS infectious agent.

1983
The infectious agent was isolated by L. Montagnier (Paris) and R. C.
Gallo (NIH). First called it HTLV – for human T-cell leukemia virus. Now
called Human Immunodeficiency Virus, or HIV.

2001
500,000 - 1,000,000 people infected in the US. More than 30 million
people infected world-wide; ~70% in Sub-Saharan Africa.
Fulfilling Koch's postulates difficult, because of the long latency period.

Dewhurst S., Ramothea L.W. da Cruz 1 & Whetter L. Frontiers in

Bioscience, 5, d30-49, January 1, 2000
 Some of the evidence that HIV causes AIDS

1. Virus can be isolated from almost all with the disease.

2. Advanced disease correlates with higher virus titer.
3. Asymptomatic individuals that have antibodies to coat
proteins later develop the disease.
4. Recipients of contaminated blood frequently develop
AIDS.
5. About 30% of children born to infected mothers are
infected with the virus; those that are, go on to
develop AIDS, but uninfected siblings do not.
6. AIDS does not appear in a new locality without the prior
presence of HIV.
7. Treatments that target the virus alleviate the disease.
Structure of HIV

Sarcophagus-shaped capsid
contains 2 copies of RNA genome
(SS (+) strand), a reverse
transcriptase, integrase, and
protease.

P7 coats the RNA, and P24 forms

the nucleocapsid structure, which
is enclosed by a lipid bilayer.

Lipid bilayer comes from the host

cell, but contains two viral-
encoded glycoproteins, gp41 (41
kDa) and gp120 (120 kDa).

gp120 binds the CD4 receptor on

helper T cells.
•  HIV is a retrovirus.

•  A more specific name is Lentivirus.

•  Retroviruses have an RNA genome that is

replicated via a DNA intermediate in infected
cells. DNA also integrates in the host genome
to form provirus.

•  HIV is more complex than RNA tumor

viruses, such as MLV, murine leukemia virus.
–  HIV encodes a number of extra regulatory and
accessory proteins.
Life Cycle
HIV’s genome is ~ 9.8 KB, and encodes 2 other classes of
proteins besides the usual GAG-POL-ENV proteins.
 HIV gene expression
is mainly from
integrated provirus
and is separated
temporally into early
genes and late
genes.

Late gene expression

requires Rev (which
moves un- and partially
spliced RNA to
cytoplasm).

RRE is the binding

site for Rev.

Unspliced RNA is
incorporated into
virions (genome).
D1-D4 are splice donor sites (5’ SS); A1-A7 are splice acceptor sites (3’ SS), > 20 RNAs are generated.
GAG & GAG-POL fusion proteins are processed by HIV Protease (part of Pol), GP160 is processed by cellular protease.
MA – matrix, CA-capsid, NC- nucleocapsid
Activating
transcription
of the HIV
genome.

The LTR promoter contains binding sites for several cellular transcription factors, including
(common) SP1 and TBP factors, but also NF-kappa B. The activity of NF-kappa B is inducible, and
is regulated by several external signals that stimulate T cells.
The HIV trans-activation response (TAR) element is an RNA element which is known to be
required for the trans-activation of the viral promoter and for virus replication. The TAR hairpin is a
dynamic structure that acts as a binding site for the Tat protein, and this interaction stimulates the
activity of the long terminal repeat promoter.
Tat binds to the TAR RNA domain, and stimulates transcription by
promoting elongation (i.e., promoter clearance) by RNAP II.
The Tat-RNA complex promotes phosphorylation of the CTD of RNAP
II by recruiting kinases that catalyze its phosphorylation.
Infection Cycle
Tat, nef and
rev genes
expressed
early.

Gag, Pol and

Env genes
expressed late.
 Infection

gp120 and gp41 associate

with one another. The
complete complex is
probably trimeric (3 copies of
each protein).

Beta turns in C3 and C4 regions are

important for binding to the cell-
surface CD4 receptor.

Primary Target: CD4 helper T cells.

The normal role of these cells is to stimulate macrophages to destroy
pathogens, and coordinate the immune response. They have on their
surface, a glycoprotein called CD4. The viral protein gp120 binds CD4.
Gp120 also binds the chemokine co-receptor. gp41 causes membrane
fusion (between virion and cell).
 gp120
(deglycosylated)

CD4

Fab fragment
of a
neutralizing
Antibody

Kwong et al. Nature 393, 648 (1998)

 Co-receptors for HIV

Chemokine receptor
family: CCR5, CXC,
CXCR4, CCR2b

CD4 is necessary but

not sufficient for HIV
entry into CD4+ T-
lymphocytes.

The chemokine
receptors act as co-
receptors.
Why is the disease less aggressive in some people?
Mutations in chemokine co-receptors that confer resistance.

CCR5 polymorphism
- About 1% of Caucasians are resistant to the virions
- 32 bp deletion in this gene (second extracellular loop)
- these people can still get HIV from variants that can use the
CXCR4 co-receptor.
CXCR4 3' UTR mutation
- ~ 1% Caucasians
- delays the disease onset and time of death
- point mutation in the 3' UTR. Mechanism unknown.

As disease progresses, get variants that use CCR3 or CCR2b;

these virions are more aggressive.
Late stages, the CXC co-receptor is used; these virions are
cytopathic and syncytium-inducing.
How does HIV kill cells?
Virus budding from cell membrane is not lethal. Cells die by
autofusion, syncytial formation, and apoptosis. Other
mechanisms?

1. Autofusion
CD4 and gp120/41
proteins mediate fusion
and intracellular vesicle
formation.
2. Syncytium formation
gp120/41 proteins on
infected cells bind to CD4
receptors on normal cells,
causing cell fusion. The
giant multi-nucleated
syncytium dies before
long.

Infected cell
Normal cell

3.Apoptosis
An infected helper T cell can direct an uninfected T helper cell to undergo
apoptosis (programmed cell death). Apoptosis can be normal, for
example, to eliminate auto-reactive T lymphocytes to establish self-
tolerance.
 Current Therapies
1.  Nucleoside analogues and other RT
inhibitors.
2.  Active site inhibitors of the HIV
protease (part of the Pol gene).
3.  Interferon (stimulates anti-viral
response)
4.  Cocktails of all 3.
 Nucleoside analogues

Inhibit at the reverse transcriptase step by causing chain termination. The

trick (to reduce toxicity) is to find one that the HIV reverse transcriptase
accepts readily, but the host's DNA polymerases tend to reject.
 Vaccines?
Whole virus vaccines
Attenuated viruses: Essentially intact, living HIV virions that have
been chemically or genetically damaged.
Whole killed virus: Intact virions that have been damaged so badly
that they are completely nonfunctional (dead).

Subunit vaccines
Clone one gene from HIV, express the protein and use it to vaccinate
patients. The disadvantage is that the person only raises antibodies
against one target. With free virus, the targets are mainly the
envelope proteins; however, these are extremely variable proteins. Six
amino acids of the V3 loop of gp120 appear to be relatively constant
(some variability exists but most antibodies cross react with the
variants). Antibodies against cocktails of different V3's are being tried.

Nucleic Acid Vaccines

Gene gun, muscle expression.
A Major Challenge in Maintaining
Control of HIV
•  HIV evolves rapidly
•  The RT is error-prone (no proof-
reading)
•  ~ 1-2 mutations in each cDNA copy of
the 9.8 kb RNA genome