Significance of Retroviruses

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Significance of Retroviruses
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Cloning of AIDS-Associated Retrovirus
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Western for Anti-HIV-1 Antibodies
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Evidence that AIDS is Caused by HIV-1
Syncytia Induction in PBMC by Cloned HIV-1
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HIV-2
Genetically similar to HIV-1
Identified in asymptomatic, HIV-1
seronegative individuals with antibodies against
SIV capsid
Causes AIDS, progression less rapid than with
HIV-1
Blood-bank ELISA detects HIV-1 and HIV-2
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HIV-1 member of large family of viruses that
co-evolved with African primates
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HIV-1 Open Reading Frames
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tat
HIV-1 Genome
vpr
-vpu
rev-
-nef
R
U5
pol
env
gag
tat-
vif
rev
R
U3
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Standard Approach to the Study of Retroviruses
Provirus cloned from genomic library,
propagated and mutated in plasmid
DNA transfection of mammalian cell lines for
virion production
Biochemical and functional analysis of virions
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(For exogenous retrovirus start here)
Maturation
Binding Membrane Fusion
Retroviral Life Cycle
Uncoating Reverse Transcription
Budding
Expression Membrane Targeting
Nuclear transport Integration
(For endogenous retrovirus start here)
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HIV-1 Membrane Fusion Machine
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Targets of Anti-HIV-1 Drugs
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Retroviral Virion Genomic RNA
Identical to full-length viral mRNA 5 m7GpppN
cap structure 3 polyA splicing signals
U unique, e.g., U5 is unique to 5 end of the
RNA genome
Cis-acting signals for replication R is a
direct linear repeat required for strand
transfer PBS primer binding site for first
strand synthesis PPT polypurine tract primes
second strand synthesis
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Overview of Reverse Transcription
Genomic RNA (virion)
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Targets of Anti-HIV-1 Drugs
Reverse Transcriptase
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Integration of Retroviral cDNA into
Host Chromosomal DNA Establishes the Provirus
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Targets of Anti-HIV-1 Drugs
Integrase
Required for viral replication, no
expression without integration
Specialized viral enzyme, thus an attractive
target for new drug
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HIV-1 U3 Enhancer Sites
U3 of the 5LTR of the provirus contains the
single viral promoter which contains
binding sites for factors found in T cells and
macrophages.
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The Paradox of HIV-1 Tat
Trans-acting transcriptional activator
Binds stem-loop structure (TAR) in nascent HIV-1
transcript
How does Tat stimulate transcription of the RNA
to which it binds?
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Tat Enhances Processivity of RNA Polymerase II
CTD
RNA pol II
CDK9
Cyclin T
RNA pol II
Tat
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How does HIV-1 express 9 orfs from 1 mRNA?
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HIV-1 expresses more than 30 mRNAs
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Use of Polyproteins Increases Retroviral Coding
Capacity
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Retroviral Protease Activation
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Molecular Targets of Anti-HIV-1 Drugs
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