Molecular Biology of HIV

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Molecular Biology of HIV

• www.hopkins-aids.edu/hiv_lifecycle/
• www.who.int/hiv/en
• hivinsite.ucsf.edu/InSite.jsp?dockb-02-01-02

HIV budding from an infected cell.
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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.
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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
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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.
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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.
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• 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.

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(No Transcript)
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Life Cycle
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HIVs genome is 9.8 KB, and encodes 2 other
classes of proteins besides the usual GAG-POL-ENV
proteins.
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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).
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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.
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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.
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Infection Cycle
Tat, nef and rev genes expressed early.
Gag, Pol and Env genes expressed late.
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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).
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gp120 (deglycosylated)
CD4
Fab fragment of a neutralizing Antibody
Kwong et al. Nature 393, 648 (1998)
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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.
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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.
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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.
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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.
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Current Therapies

• Nucleoside analogues and other RT inhibitors.
• Active site inhibitors of the HIV protease (part
  of the Pol gene).
• Interferon (stimulates anti-viral response)
• Cocktails of all 3.

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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.
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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.
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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