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 1 st 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 cloned using genomic library from infected cell Lymph node cells co-cultivated with uninfected lymphocytes RT activity transferred to fresh lymphocytes tat-gag RU5 polenv rev tat vpr rev--nef -vpu vif R U3 Cloning of AIDS-Associated Retrovirus

HIV-1 Serology p24 ELISA Western Blot screen for anti-Capsid antibody highly sensitive virion proteins separated by SDS-PAGE highly specific gag polenv rev tat vpr -nef rev- -vpu tat-vif RU5 R U3 MA CA NC p6

Microtitre well coated with p24 Serum added Anti-human antibody labeled with enzyme added Enzyme substrate added Enyzme-linked immunosorbent assay to 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 after HIV-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 individuals and 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 cloned HIV-1 in Chimps and even 1 human

HIV-2 Identified in asymptomatic, HIV-1 seronegative individuals with antibodies against SIV capsid Genetically similar to HIV-1 Causes AIDS, progression less rapid than with HIV-1 Blood-bank ELISA detects HIV-1 and HIV-2

HIV-1: member of large family of viruses that co-evolved with African primates 1. Each virus adapted to particular primate species 2. Disease occurs when viruses jump to non-native host: - SIV SM from Sooty mangabey to macaque - HIV-2 is identical to SIV SM - HIV-1 is identical to virus in feral chimps

HIV-1 Open Reading Frames tat-gag RU5 polenv rev tat vpr rev- -nef -vpu vif R U3 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- RU5 rev tat vpr rev--nef -vpu vif R U3 HIV-1 Genome Reverse Transcriptase Integrase Lipid Bilayer Matrix, p17 Capsid, p24 Nucleocapsid, p7 Genomic RNA Surface glycoprotein (gp120) Transmembrane glycoprotein (gp41) An HIV-1 Virion pol gag env

DNA transfection of mammalian cell lines for virion production Biochemical and functional analysis of virions Provirus cloned from genomic library, propagated and mutated in plasmid Standard Approach to the Study of Retroviruses

Expression Membrane Targeting Budding Maturation Nuclear transport Integration Uncoating Reverse Transcription Binding Membrane Fusion Retroviral Life Cycle (For exogenous retrovirus start here) (For endogenous retrovirus start here)

HIV-1 Membrane Fusion Machine CD4 Chemokine Receptor Target Cell Membrane gp120 gp41 Virion Membrane 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 interaction Soluble CD4 Disruption of the gp120-CKR interaction High chemokines in long-term non-progressors Mutant CKR in exposed, uninfected individuals Stabilization of Pre-Hairpin intermediate Soluble C-peptide or analogues

Retroviral Virion Genomic RNA RU5U3R gag envpol SD SA PBSPPT 5'Cap A n Identical to full-length viral mRNA: 5’ m 7 GpppN cap structure 3’ polyA splicing signals 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 U: unique, e.g., U5 is unique to 5’ end of the RNA genome

Overview of Reverse Transcription Genomic RNA (virion) RU5U3R gag envpol SD SA PBSPPT 5'Cap A n RU5U3R env U5U3 5’ LTR polgag 3’ 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 analogues Nonnucleoside inhibitors Combination therapy necessitated by viral resistance (RT mutations) and drug-toxicity to host

Integration of Retroviral cDNA into Host Chromosomal DNA Establishes the Provirus Nuclear MembraneChromosomal DNA Viral cDNA Provirus 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 concerted reaction 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 an attractive target for new drug Required for viral replication, no expression without integration

RU5 TATA C/EBP; Macrophages NFkB/NFAT; activated T Cells SP1; relatively general HIV-1 U3 Enhancer Sites U3 of the 5’LTR of the provirus contains the single viral promoter which contains binding sites for factors found in T cells and 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? TAR Tat Flanking chromosomal DNA U3RU5

Tat Enhances Processivity of RNA Polymerase II U3RU5 RNA pol II PPPP TAR CDK9 Cyclin T Tat RNA pol II CTD 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 RU5 polenv rev tat vpr rev- -nef -vpu vif R U3 One promoter (5’U3) makes one primary transcript Complex splicing, ribosomal frame-shift, polyproteins Proteins translated in accordance with ribosomal scanning model

tat-gag polenv rev tat vpr rev- -nef -vpu vif Acceptors: Donors: HIV-1 expresses more than 30 mRNAs How do unspliced or singly-spliced mRNAs exit the nucleus despite 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 NES RanGTP Nuclear Membrane SD RRE U5R SA U3R

Use of Polyproteins Increases Retroviral Coding Capacity tat-gag RU5 polenv rev tat vpr rev- -nef -vpu vif R U3 The primary translation products of gag, pol, and env are all expressed as polyproteins that are post-translationally cleaved into multiple products. The Gag and Gag-Pol Polyproteins are cleaved by the pol-encoded Protease at the time of virion assembly The Env polyprotein is cleaved by a cellular protease in the Golgi

The Gag Polyprotein Targets Virion Assembly to the Plasma Membrane and is Sufficient for Release of Virion Particles Gag Polyprotein Plasma Membrane Immature Virion

Retroviral Protease Activation

Molecular Targets of Anti-HIV-1 Drugs Viral Protease Inhibitors of this enzyme are now a standard part of 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 effectively inhibits viral replication without appearance of drug-resistant virus Persistent reservoirs of viral infection include long-lived memory T-cells bearing proviral DNA and macrophages

New York, Feb 15th, 2003 Vote as if your grant life depended on it