Significance of Retroviruses

1. Defiance of the Central Dogma Model Mobile Genetic Elements Discovery of Oncogenes Vectors for cDNA delivery Disease Causing Agents Significance of Retroviruses

2. AIDS: A New Disease? 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...

3. AIDS: Clues to a Retroviral Etiology Blood-borne transmissible agent: HiVolSA, IVDU, Hemophilia CD4+ T-cells reduced with AIDS 1st human retrovirus (1980), CD4-tropic Human T Cell Leukemia Virus Type 1 Immune defects with animal retroviruses

4. Lymph node cells co-cultivated with uninfected lymphocytes Reverse transcriptase detected in supernatant RT activity transferred to fresh lymphocytes tat vpr -vpu rev- -nef Virions detected by electron microscopy R U5 pol env Infectious 9.8 kB DNA provirus cloned using genomic library from infected cell gag vif tat- rev R U3 Cloning of AIDS-Associated Retrovirus

5. HIV-1 Serology p24 ELISA screen for anti-Capsid antibody highly sensitive p6 MA CA NC p6 Western Blot virion proteins separated by SDS-PAGE highly specific tat vpr rev- -vpu -nef R U5 pol env gag vif tat- rev R U3

6. Enyzme-linked immunosorbent assay to detect anti-HIV-1 p24 antibodies Microtitre well coated with p24 Serum added Anti-human antibody labeled with enzyme added Enzyme substrate added

7. Lyse HIV-1 in detergent -gp160 -gp120 -p66 Separate proteins by SDS-PAGE -p55 -pg41 Transfer proteins to membrane -p32 -p24 Cut membrane and incubate in sera -p17 Detect bound antibodies 6 30 Days after HIV-1 infection Western for Anti-HIV-1 Antibodies

8. 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 Evidence that AIDS is Caused by HIV-1 Syncytia Induction in PBMC by Cloned HIV-1

9. 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

10. 1. Each virus adapted to particular primate species 2. Disease occurs when viruses jump to 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: member of large family of viruses that co-evolved with African primates

11. tat vpr -vpu rev- -nef R U5 pol env gag vif tat- rev gag and env: virion structural proteins R U3 pol: Protease, RT, and Integrase vif, vpr, vpu, and nef:non-essential accessory genes tat and rev: essential accessory genes HIV-1 Open Reading Frames

12. HIV-1 Virion Reverse Transcriptase Integrase Lipid Bilayer Genomic RNA Matrix, p17 Capsid, p24 Nucleocapsid, p7 An An Transmembrane glycoprotein (gp41) Surface glycoprotein (gp120) HIV-1 Genome tat vpr -vpu rev- -nef R U5 pol env gag vif tat- rev R U3

13. 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

14. (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)

15. Fusion peptide CD4 C N gp120 C N Chemokine Receptor gp41 Virion Membrane Target Cell Membrane HIV-1 Membrane Fusion Machine

16. 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

17. 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 Targets of Anti-HIV-1 Drugs

18. SD SA gag pol env A R U5 U3 R 5'Cap n PBS PPT 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

19. SD SA gag pol env A R U5 U3 R 5'Cap n PBS PPT Occurs in target cell cytoplasm within a complex of viral proteins Viral cDNA Template: viral genomic RNA; Primer: tRNA annealed to PBS in virion gag pol env U3 R U5 U3 R U5 Reverse Transcriptase: viral DNA polymerase that utilizes RNA or DNA as template; also has RNaseH activity (degrades RNA when complexed with DNA). 5’ LTR 3’ LTR Product is longer than the template, due to two “jumps” Overview of Reverse Transcription Genomic RNA (virion)

20. Major target of anti-viral drugs in clinical use Combination therapy necessitated by viral resistance (RT mutations) and drug-toxicity to host Two main classes of inhibitors: Nucleoside analogues Nonnucleoside inhibitors Targets of Anti-HIV-1 Drugs Reverse Transcriptase

21. Viral cDNA Provirus Integration Integration: covalent linkage of ds viral cDNA to host chromosomal DNA. Site of integration is random with respect to host chromosomal DNA. Provirus: permanent genetic element in the infected cell, and in all of the cell’s progeny Nuclear Membrane Chromosomal DNA 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. Integration of Retroviral cDNA into Host Chromosomal DNA Establishes the Provirus

22. 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

23. 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 provirus contains the single viral promoter which contains binding sites for factors found in T cells and macrophages.

24. Tat TAR U3 R U5 Flanking chromosomal DNA 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?

25. TAR P P P P U3 R U5 In the absence of Tat: transcription initiates at normal rate transcripts are short, not full-length Tat stimulates transcriptional elongation Tat Enhances Processivity of RNA Polymerase II CTD RNA pol II CDK9 Cyclin T RNA pol II Tat

26. tat vpr -vpu rev- -nef R U5 pol env gag vif tat- rev One promoter (5’U3) makes one primary transcript R U3 Proteins translated in accordance with ribosomal scanning model Complex splicing, ribosomal frame-shift, polyproteins How does HIV-1 express 9 orfs from 1 mRNA?

27. Acceptors: tat vpr -vpu rev- -nef pol env gag vif tat- rev Donors: Multiply spliced mRNAs readily exit the nucleus How do unspliced or singly-spliced mRNAs exit the nucleus despite presence of multiple splicing signals? HIV-1 expresses more than 30 mRNAs

28. Nuclear Export of Unspliced HIV-1 RNA by Rev Nuclear Membrane CRM1 RanGTP NES REV RRE U3 R R U5 SA SD 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

29. tat vpr -vpu rev- -nef R U5 pol env gag vif tat- rev The primary translation products of gag, pol, and env are all expressed as polyproteins that are post-translationally cleaved into multiple products. R U3 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 Use of Polyproteins Increases Retroviral Coding Capacity

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

31. Retroviral Protease Activation

32. Viral Protease Not required for virion assembly Required for virion maturation and infectivity Inhibitors of this enzyme are now a standard part of the anti-HIV drug regimen Combination therapy necessitated by resistance Molecular Targets of Anti-HIV-1 Drugs

33. 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

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