CROI2104 CONFERENCE ON RETROVIRUSES AND OPPORTUNISTIC INFECTIONS
Significance of Retroviruses
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Transcript of Significance of Retroviruses
Significance of Retroviruses
Defiance of the Central Dogma
Model Mobile Genetic Elements
Discovery of Oncogenes
Vectors for cDNA delivery
Disease Causing Agents
Morbidity and Mortality Weekly Report
June 5, 1981
Pneumocystis Pneumonia --- Los Angeles
In the period October 1980-May 1981, 5 young men, all active homosexuals, were treated for biopsy-confirmed Pneumocystis carinii pneumonia at 3 different hospitals in Los Angeles, California. Two of the patients died...
AIDS: A New Disease?
AIDS: Clues to a Retroviral Etiology
Blood-borne transmissible agent:HiVolSA, IVDU, Hemophilia
CD4+ T-cells reduced with AIDS
Immune defects with animal retroviruses
1st human retrovirus (1980), CD4-tropic Human T Cell Leukemia Virus Type 1
Reverse transcriptase detected in supernatant
Virions detected by electron microscopy
Infectious 9.8 kB DNA provirus clonedusing genomic library from infected cell
Lymph node cells co-cultivated with uninfected lymphocytes
RT activity transferred to fresh lymphocytes
tat-gag
R U5pol env
rev
tatvpr
rev- -nef-vpu
vif
RU3
Cloning of AIDS-Associated Retrovirus
HIV-1 Serologyp24 ELISA
Western Blot
screen for anti-Capsid antibodyhighly sensitive
virion proteins separated by SDS-PAGEhighly specific
gagpol env
rev
tatvpr
-nefrev-
-vpu
tat-vif
R U5
RU3
MA CA NC p6p6
Microtitrewell
coatedwithp24
Serumadded
Anti-humanantibody
labeled withenzymeadded
Enzymesubstrate
added
Enyzme-linked immunosorbent assayto detect anti-HIV-1 p24 antibodies
Lyse HIV-1 in detergent
Separate proteins by SDS-PAGE
Transfer proteins to membrane
Cut membrane and incubate in sera
Detect bound antibodies
Western for Anti-HIV-1 Antibodies
-gp160
-p66-p55
-p24
-pg41
-p32
-p17
-gp120
Days afterHIV-1 infection
306
Evidence that AIDS is Caused by HIV-1
Syncytia Induction in PBMC by Cloned HIV-1
HIV-1-seropositivity predicts AIDS in individualsand in populations (anticipates spreading pandemic)
Cloned HIV-1 infects CD4+ T cells, dendritic cells, macrophages
SCID-hu mouse model recapitulates AIDS pathology
SIV/macaque model with similar immunodeficiency
Koch’s Postulates satisfied with clonedHIV-1 in Chimps and even 1 human
HIV-2
Identified in asymptomatic, HIV-1 seronegativeindividuals with antibodies against SIV capsid
Genetically similar to HIV-1
Causes AIDS, progression less rapid thanwith HIV-1
Blood-bank ELISA detects HIV-1 and HIV-2
HIV-1: member of large family of virusesthat co-evolved with African primates
1. Each virus adapted to particularprimate species
2. Disease occurs when viruses jumpto non-native host:
- SIVSM from Sooty mangabey to macaque
- HIV-2 is identical to SIVSM
- HIV-1 is identical to virus in feral chimps
HIV-1 Open Reading Frames
tat-gag
R U5pol env
rev
tatvpr
rev- -nef-vpu
vif
RU3
gag and env: virion structural proteins
pol: Protease, RT, and Integrase
vif, vpr, vpu, and nef: non-essential accessory genes
tat and rev: essential accessory genes
tat-
R U5
rev
tatvpr
rev- -nef-vpu
vif
RU3
HIV-1 Genome
ReverseTranscriptase
Integrase Lipid Bilayer
Matrix, p17
Capsid, p24
Nucleocapsid, p7
GenomicRNA
Surfaceglycoprotein (gp120)
Transmembraneglycoprotein (gp41)
An An
HIV-1 Virion
polgag
env
DNA transfection of mammalian cell linesfor virion production
Biochemical and functionalanalysis of virions
Provirus cloned fromgenomic library, propagatedand mutated in plasmid
Standard Approach to the Study of Retroviruses
ExpressionMembrane Targeting
Budding
Maturation
Nuclear transportIntegration
UncoatingReverse Transcription
BindingMembrane Fusion
RetroviralLife
Cycle
(For exogenous retrovirus start here)
(For endogenous retrovirus start here)
HIV-1 Membrane Fusion Machine
CD4
ChemokineReceptor
TargetCell
Membrane
gp120
gp41
VirionMembrane
Fusion peptide
NC
NC
Chemokine Receptors and HIV-1
CCR5 and CXCR4 are the two main ones used by HIV-1
Usage determined by sequences on gp120 V3 loop
CCR5 using viruses are transmitted preferentially
CXCR4 using viruses usually appear late, not always
Macrophages and Dendritic cells only infected by CCR5 viruses
Primary T cells may express either, T cell lines only CXCR4
CXCR4-using viruses are more aggressive and cause synctia
Targets of Anti-HIV-1 Drugs
Disruption of the gp120-CD4 interactionSoluble CD4
Disruption of the gp120-CKR interactionHigh chemokines in long-term non-progressors
Mutant CKR in exposed, uninfected individuals
Stabilization of Pre-Hairpin intermediateSoluble C-peptide or analogues
Retroviral Virion Genomic RNA
R U5 U3 Rgag envpolSD SA
PBS PPT
5'Cap An
Identical to full-length viral mRNA:5’ m7GpppN cap structure3’ polyAsplicing signals
Cis-acting signals for replication:R is a direct linear repeat required for strand transferPBS: primer binding site for first strand synthesisPPT: polypurine tract primes second strand synthesis
U: unique, e.g., U5 is unique to 5’ end of the RNA genome
Overview of Reverse Transcription
Genomic RNA (virion)
R U5 U3 Rgag envpolSD SA
PBS PPT
5'Cap An
R U5 U3 Renv U5U35’ LTR
polgag3’ LTR
Viral cDNA
Occurs in target cell cytoplasm within a complex of viral proteins
Product is longer than the template, due to two “jumps”
Template: viral genomic RNA; Primer: tRNA annealed to PBS in virion
Reverse Transcriptase: viral DNA polymerase that utilizes RNA or DNA as template;also has RNaseH activity (degrades RNA when complexed with DNA).
Targets of Anti-HIV-1 Drugs
Reverse Transcriptase
Major target of anti-viral drugs in clinical use
Two main classes of inhibitors:Nucleoside analoguesNonnucleoside inhibitors
Combination therapy necessitated by viralresistance (RT mutations) anddrug-toxicity to host
Integration of Retroviral cDNA into HostChromosomal DNA Establishes the Provirus
Nuclear Membrane Chromosomal DNA
Viral cDNAProvirus
Integration
Integration: covalent linkage of ds viral cDNA to host chromosomal DNA.
Provirus: permanent genetic element in the infected cell, and in all of the cell’s progeny
Integrase: viral nuclease that clips 2 nucleotides from the 3’ end of the 2 LTRs. In a concertedreaction it then makes a staggered cut in host chromosomal DNA, and ligates the clipped 3’ends of viral DNA to host DNA.
Site of integration is random with respect to host chromosomal DNA.
Targets of Anti-HIV-1 Drugs
Integrase
Specialized viral enzyme, thus anattractive target for new drug
Required for viral replication, no expressionwithout integration
R U5
TATA
C/EBP; Macrophages
NFkB/NFAT; activated T Cells
SP1; relatively general
HIV-1 U3 Enhancer Sites
U3 of the 5’LTR of the proviruscontains the single viral promoterwhich contains bindingsites for factors found in T cellsand macrophages.
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?
TARTat
Flanking chromosomal DNA
U3 R U5
Tat Enhances Processivity of RNA Polymerase II
U3 R U5
RNA pol IIP P PP
TAR
CDK9
Cyclin T
Tat
RNA pol IICTD
Tat stimulates transcriptional elongation
In the absence of Tat:
transcription initiates at normal rate
transcripts are short, not full-length
How does HIV-1 express 9 orfs from 1 mRNA?
tat-gag
R U5pol env
rev
tatvpr
rev- -nef-vpu
vif
RU3
One promoter (5’U3) makes one primary transcript
Complex splicing, ribosomal frame-shift, polyproteins
Proteins translated in accordance with ribosomal scanning model
tat-gagpol env
rev
tatvpr
rev- -nef-vpu
vif
Acceptors:
Donors:
HIV-1 expresses more than 30 mRNAs
How do unspliced or singly-spliced mRNAs exit the nucleusdespite presence of multiple splicing signals?
Multiply spliced mRNAs readily exit the nucleus
Nuclear Export of Unspliced HIV-1 RNA by Rev
Rev binds Rev Response Element (RRE), stem-loop in unspliced RNA
Rev NES (LPPLERLTL) recognized by CRM1
CRM1/RanGTP targets Rev and bound RNA to the nuclear pore
CRM1
REV
NESRanGTP
Nuclear Membrane
SD
RREU5R
SAU3 R
Use of Polyproteins Increases Retroviral Coding Capacity
tat-gag
R U5pol env
rev
tatvpr
rev- -nef-vpu
vif
RU3
The primary translation products of gag, pol, and env are all expressed aspolyproteins that are post-translationally cleaved into multiple products.
The Gag and Gag-Pol Polyproteins are cleaved by the pol-encoded Proteaseat the time of virion assembly
The Env polyprotein is cleaved by a cellular protease in the Golgi
The Gag Polyprotein Targets VirionAssembly to the Plasma Membrane and isSufficient for Release of Virion Particles
Gag Polyprotein
Plasma Membrane
Immature Virion
Retroviral Protease Activation
Gag Polyprotein
Gag-Pol Polyprotein
Plasma MembraneInduction of virion bud at the cell surface
Dimerization of Gag-Pol PolyproteinActivates ProteaseFormation ofMature HIV-1Virion
Immature VirionMature Virion
Molecular Targets of Anti-HIV-1 Drugs
Viral Protease
Inhibitors of this enzyme are now a standard partof the anti-HIV drug regimen
Not required for virion assembly
Required for virion maturation and infectivity
Combination therapy necessitated by resistance
Is Cure of HIV Infection Possible?
Combination anti-viral therapy effectivelyinhibits viral replication without appearanceof drug-resistant virus
Persistent reservoirs of viral infection includelong-lived memory T-cells bearing proviralDNA and macrophages
New York, Feb 15th, 2003
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