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HIV Structure, Lifecycle and Replication (1) Background: Basic Virology and Pathogenesis

HIV Structure, Lifecycle and Replication (1) Background: Basic Virology and Pathogenesis Structure: Virion structure, genomic structure, and accessory molecules Lifecycle: Infection and Expression January 9 & January 14 2013 F. Javier Ibarrondo, Ph.D. fibarrondo@mednet.ucla.edu.

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HIV Structure, Lifecycle and Replication (1) Background: Basic Virology and Pathogenesis

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  1. HIV Structure, Lifecycle and Replication (1) • Background: Basic Virology and Pathogenesis • Structure: Virion structure, genomic structure, • and accessory molecules • Lifecycle: Infection and Expression • January 9 & January 14 2013 • F. Javier Ibarrondo, Ph.D. • fibarrondo@mednet.ucla.edu

  2. (I) Identification of AIDS Pneumocystis pneumonia—Los Angeles. Gottlieb M S, Schanker H M, Fan P T, Saxon A, Weisman J D & Polzalski “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. All 5 patients had laboratoryconfirmed previous or current cytomegalovirus (CMV) infection and candidal mucosal infection”. Morbid. Mortal, Weekly Rep. 30:250-2. 1981. Pneumocystis carinii Pneumonia and Mucosal Candidiasis in Previously Healthy Homosexual Men — Evidence of a New Acquired Cellular Immunodeficiency Michael S. Gottlieb, M.D., Robert Schroff, Ph.D., Howard M. Schanker, M.D., Joel D. Weisman, D.O., Peng Thim Fan, M.D., Robert A. Wolf, M.D., and Andrew Saxon, M.D. N Engl J Med 1981; 305:1425-1431December 10, 1981 (II) Isolation of the virus Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS) F. Barré-Sinoussi; J. C. Chermann; F. Rey; M. T. Nugeyre; S. Chamaret; J. Gruest; C. Dauguet; C. Axler-Blin; F. Vézinet-Brun; C. Rouzioux; W. Rozenbaum; L. Montagnier. (May 20, 1983) Science, New Series, Vol. 220, No. 4599., pp. 868-871 Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS Gallo RC. et al. (May 1984) Science 224 (4648): 497–508 (III) Link Virus-AIDS

  3. From GRID to AIDS NEW HOMOSEXUAL DISORDER WORRIES HEALTH OFFICIALS By LAWRENCE K. ALTMAN. Published: May 11, 1982 Pneumocystis carinii Pneumonia and Mucosal Candidiasis in Previously Healthy Homosexual Men — Evidence of a New Acquired Cellular Immunodeficiency Michael S. Gottlieb, M.D., Robert Schroff, Ph.D., Howard M. Schanker, M.D., Joel D. Weisman, D.O., Peng Thim Fan, M.D., Robert A. Wolf, M.D., and Andrew Saxon, M.D. N Engl J Med 1981; 305:1425-1431December 10, 1981 An African HIV-1 sequence From 1959 and implications for the origin of the epidemic Tuofu Zhu, Bette T. Korber, Andre J. Nahmiask, Edward Hooper, Paul M. Sharp & David D. Ho. February 1998 Nature, Vol 391, 5 The emergence of HIV/AIDS in the Americas and beyond M. Thomas P. Gilbert, Andrew Rambaut, Gabriela Wlasiuk, Thomas J. Spira, Arthur E. Pitchenik, and Michael Worobey PNAS , November 20, 2007, 18566–18570

  4. Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). Since the beginning of the epidemic in 1981, more than 60 million people have contracted HIV and nearly 30 million have died of HIV-related causes. At the end of 2011, an estimated 34 million people, an estimated 0.8% of adults aged 15-49 years worldwide, are living with HIV. 2.5 million new infections in 2011; 330,000 were children. 7,000 people contract HIV everyday, nearly 300 every hour. In 2011 alone, AIDS claimed an estimated 1.7 million lives, of which 230,000 were children. HIV primarily infects vital cells in the human immune system such as helper T cells (CD4+ T cells), macrophages and dendritic cells. HIV infection leads to low levels of CD4+ T cells. http://www.unaids.org/en/

  5. Viruses Microscopic infectious agents that can infect the cells of a biological organism. Viruses can only replicate themselves by infecting a host cell and are incapable to reproduce on their own. A complete viral particle, known as a virion consists of nucleic acid surrounded by a protective coat of protein called a capsid.

  6. Types of Viruses (Baltimore Classification) I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) Herpesviridae (Herpes, CMV, EBV), Poxviridae (Smallpox, Chickenpox, Vaccinia), Papilloma virus, Adenovirus II: Single-stranded (+) sense DNA (Parvoviruses) Erythema infectiosum, Phages III: Double-stranded RNA (Reoviruses; Birnaviruses)Rotavirus, Reovirus IV: Single-stranded (+) sense RNA (Picornaviruses; Togaviruses, etc)Polio, SARS, Hep A, Hep C, Rubella, Yellow fever V: Single-stranded (-) sense RNA (Orthomyxoviruses, Rhabdoviruses, etc)Rubella,Influenza, Rabies, Measles, Mumps, Ebola VI: Single-stranded (+) sense RNA with DNA intermediate (Retroviruses)HTLV,HIV VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses) Hep B

  7. HIV Classification Group: Group VI (ssRNA-RT) Family: Retroviridae Genus: Lentivirus(Enveloped) Species: Human immunodeficiency virus 1 Species: Human immunodeficiency virus 2 Species Virulence Infectivity Prevalence Inferred origin HIV-1 High High Global Chimpanzee HIV-2 Lower Low West Africa Sooty Mangabey

  8. HIV phylogeny CRF14_BG

  9. HIV-1 Tropism www.hivmedicine.com/textbook/pathogen.htm

  10. HIV-1 Transmission Sexual route. Blood or blood product route. Mother-to-child transmission (MTCT).

  11. Viral RNA gp120 p24 RT & other virion proteins Reverse transcription Infection Fusion & Entry Binding Nuclear localization & entry Integration CD4 CXCR4

  12. Viral RNA gp120 p24 RT & other virion prteins Viral Gene Transcription Cellular Activation Assembly Post-translational processing Budding Translation Expression

  13. HIV Structure • Virion • Genomic • Proteomic

  14. HIV Structure SIV HIV

  15. HIV Structure • Virion • Genomic • Proteomic

  16. Viral RNA gp120 p24 RT & other virion proteins Reverse transcription Infection Fusion & Entry Binding Nuclear localization & entry Integration CD4 CXCR4

  17. HIV Genome R U5 U3 R R U5 U3 HIV RNA R U5 U3 Reverse transcription Integration HIV DNA Host DNA Host DNA LTR LTR

  18. HIV-1 RNA R U5 U3 R Nature Reviews Microbiology 2, 461-472 (June 2004) Nature 460, 711-716 (6 August 2009)

  19. HIV-1 Integrated DNA R U5 U3 R U5 U3 Host DNA Host DNA LTR LTR Source: The AmFAR AIDS Handbook, D Ward, pp. 348

  20. RNA Splicing Translational Frameshift

  21. Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19

  22. Source: Atlas of Infectious Diseases, Mandell & Mildvan (ed.), pp. 3.13

  23. Dr. Isabelle BOUALLAGA Institut Pasteur. http://www.123bio.net/

  24. Source: Atlas of Infectious Diseases, Mandell & Mildvan (ed.), pp. 3.13

  25. HIV Structure • Virion • Genomic • Proteomic

  26. HIV Proteins Structural Proteins Gag: Matrix, Capsid, NC, p6 Pol: Protease, Reverse Transcriptase, Integrase Env: gp120, gp41 Regulatory Proteins Tat Rev Nef Accessory proteins Vif Vpr Vpu

  27. Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19

  28. Source: BioAfrica Bioinformatics for HIV Research. http://bioafrica.mrc.ac.za/

  29. Gene/Protein Mass, KDa Function gag (Pr) 55gag p55 Gag precursor protein MA - Matrix p17 Aids nuclear import and viral assembly CA - Capsid p24 HIV central core – contains HIV genome and enzymes NC - Nucleocapsid p15 (p6,p9) p6 – Precise location in virion unknown, not generally present in other retroviruses, may direct proteins to secretory path of cell. p9 – Assoc. with HIV RNA, involved in packaging of HIV RNA into virions RNA binding RNA packing Association with membrane Formation of Gag multimers myristate Matrix p17 Capsid p24 p2 NC p9 p1 p6 Envelope Incorporation Viral Budding Viral Entry

  30. Source: BioAfrica Bioinformatics for HIV Research. http://bioafrica.mrc.ac.za/

  31. Gene/Protein Mass, KDa Function pol (Pr) 160gag-pol p160 Gag- Pol precursor protein PR - protease p10 Cleaves Gag and Gag-Pol IN - integrase p32 Integrates viral genome into host DNA RT – reverse p66/p51 p66 – copies RNA genome. RNAse H transcriptase p51 – regulatory? Gag PR p10 p6 RT IN p32 p51 p66

  32. Anti-HIV drugs Nucleoside/Nucleotide Reverse Transcriptase Inhibitors lamivudine (3TC), zalcitabine (ddC), zidovudine (AZT), didanosine (ddI), stavudine (d4T), tenofovir  Non-Nucleoside Reverse Transcriptase Inhibitors Delavirdine, efavirenz, nevirapine Protease Inhibitors Amprenavir , indinavir, saquinavir, saquinavir, lopinavir/ritonavir, ritonavir, nelfinavir  Integrase Inhibitors Isentress (Raltegravir or MK-0518) , JTK303/GS-9137 Fusion or Entry Inhibitors Enfurvitide (Fuzeon or T20), Maraviroc (Selzentry -CCR5 antagonist-) HAART (Highly Active Antiretroviral Therapy): three or more anti-HIV drugs (antiretrovirals) from different classes in combination allows them to work together to keep HIV levels down

  33. Gene/Protein Mass, KDa Function env (Pr) 160 env gp160 Env precursor protein SU – Envelope gp120 Surface glycoprotein that binds CD4 TM - Transmembrane gp41 Transmembrane protein that anchors gp120 to virus, responsible for fusion between virus and host cell membrane.

  34. Gene/Protein Mass, KDa Function gag (Pr) 55gag p55 Gag precursor protein MA - Matrix p17 Aids nuclear import and viral assembly CA - Capsid p24 HIV central core – contains HIV genome and enzymes NC - Nucleocapsid p15 (p6,p9) p6 – Precise location in virion unknown, not generally present in other retroviruses, may direct proteins to secretory path of cell. p9 – Assoc. with HIV RNA, involved in packaging of HIV RNA into virions pol (Pr) 160gag-pol p160 Gag- Pol precursor protein PR - protease p10 Cleaves Gag and Gag- Pol precursor proteins IN - integrase p31 Integrates viral genome into host DNA RT – reverse p66/p51 p66 – copies RNA genome. RNAse H transcriptase p51 – regulatory? env (Pr) 160 env gp160 Env precursor protein SU – Envelope gp120 Surface glycoprotein that binds CD4 TM - Transmembrane gp41 Transmembrane protein that anchors gp120 to virus, responsible for fusion between virus and host cell membrane.

  35. Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19

  36. Regulatory Proteins: TAT: Trans-Activator of Transcription REV: Regulator of Virion protein expression NEF: Negative Regulatory Factor Accessory Proteins: VIF: Virion Infectivity Factor VPU: Viral Protein U VPR: Viral Protein R

  37. TAT: Trans-Activator of Transcription TAR Tat Tat Tat Tat Tat CDK9 Recruitment of CDK9 Poor transcription Enhanced transcription Release to extracellular medium: Apoptosis bystander T-cells CXCR4 negative interaction

  38. REV: Regulator of Virion protein expression RRE = rev-response element

  39. NEF: Negative Regulatory Factor * Downmodulation the expression of several surface molecules important in host immune function: MHC I, MHC II, CD4 * Activation from latency? Extracellular Nef might activate NF kB. T-cell activation (activates the production of MIP-1alpha and MIP-1beta in macrophages)

  40. VIF: Virion Infectivity Factor www.hivmedicine.com/textbook/pathogen.htm

  41. VPU: Viral Protein U * Involved in viral budding, enhancing virion release from the cell * Downregulation of CD4 VPR: Viral Protein R * Regulation of nuclear import of the HIV-1 pre-integration complex * Required for virus replication in non-dividing cells such as macrophages. * Induces cell cycle arrest and apoptosis in proliferating cells

  42. Take-home points: • Structure: • env is only exposed viral protein (neutralization resistant) • infectivity mediated by gp120 & gp41 (CD4 & CCR5 or CXCR4) • RNA genome -- requires HIV reverse transcription to DNA • integration requires HIV integrase • LTR/promoter requires cellular transcription factors • productive viral gene expression requires HIV protease activity • accessory genes modulate: • 1) cellular function (e.g., nef, vpr) • 2) viral gene expression (e.g., tat, rev) • gag (p24) represents the primary structural component of virion • small molecule inhibitors of these structure’s functional activity • represent the primary current antiviral therapeutic strategy

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