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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