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 F. Javier Ibarrondo, Ph.D.

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: 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: December 10, 1981 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 , pp 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 (I) Identification of AIDS (II) Isolation of the virus (III) Link Virus-AIDS

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 NEW HOMOSEXUAL DISORDER WORRIES HEALTH OFFICIALS By LAWRENCE K. ALTMAN. Published: May 11, 1982 From GRID to AIDS 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 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: December 10, 1981

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 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.

Region (lower- and middle-income countries) Antiretroviral therapy coverage Estimated number of people receiving antiretroviral therapy Estimated number of people needing antiretroviral therapy Sub-Saharan Africa37%3,911,000 10,600,000 Eastern and Southern Africa41%3,203,0007,700,000 Western and Central Africa25%709,0002,900,000 Latin America and the Caribbean50%478,000950,000 Latin America51%425,000840,000 The Caribbean48%52,400110,000 East, South and South-East Asia31%739,0002,400,000 Europe and Central Asia19%114,000610,000 North Africa and the Middle East11%12,000100,000 Total36%5,254,00014,600,000

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.

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

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

CRF14_BG HIV phylogeny

HIV-1 Tropism

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

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

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

HIV Structure Virion Genomic Proteomic

HIV Structure SIVHIV

HIV Structure Virion Genomic Proteomic

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

RU5RU3 HIV RNA HIV DNA RU5 U3 RU5 U3 LTR Host DNA Reverse transcription Integration HIV Genome

Nature Reviews Microbiology 2, (June 2004) Nature 460, (6 August 2009) HIV-1 RNA RU5RU3

Source: The AmFAR AIDS Handbook, D Ward, pp. 348 HIV-1 Integrated DNA RU5 U3 RU5 U3 LTR Host DNA

RNA Splicing Translational Frameshift

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

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

Dr. Isabelle BOUALLAGA Institut Pasteur.

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

HIV Structure Virion Genomic Proteomic

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

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

Source: BioAfrica Bioinformatics for HIV Research.

Gene/ProteinMass,KDaFunction gag (Pr) 55gagp55Gag precursor protein MA - Matrixp17Aids nuclear import and viral assembly CA - Capsidp24HIV central core – contains HIV genome and enzymes NC - Nucleocapsidp15 (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 Matrix p17Capsid p24NC p9p6p1p2 myristate Association with membrane Formation of Gag multimers RNA binding RNA packing Viral Entry Envelope Incorporation Viral Budding

Source: BioAfrica Bioinformatics for HIV Research.

PR p10 RTIN p32p6 Gag p51 p66 pol (Pr) 160gag-polp160Gag-Pol precursor protein PR - proteasep10Cleaves Gag and Gag-Pol IN -integrasep32Integrates viral genome into host DNA RT – reverse transcriptase p66/p51p66 – copies RNA genome. RNAse H p51 – regulatory? Gene/ProteinMass,KDaFunction

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

env (Pr) 160 envgp160Env precursor protein SU – Envelopegp120Surface glycoprotein that binds CD4 TM - Transmembranegp41Transmembrane protein that anchors gp120 to virus, responsible for fusion between virus and host cell membrane. Gene/ProteinMass,KDaFunction

Gene/ProteinMass,KDaFunction gag (Pr) 55gagp55Gag precursor protein MA - Matrixp17Aids nuclear import and viral assembly CA - Capsidp24HIV central core – contains HIV genome and enzymes NC - Nucleocapsidp15 (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-polp160Gag-Pol precursor protein PR - proteasep10Cleaves Gag and Gag-Pol precursor proteins IN -integrasep31Integrates viral genome into host DNA RT – reverse transcriptase p66/p51p66 – copies RNA genome. RNAse H p51 – regulatory? env (Pr) 160 envgp160Env precursor protein SU – Envelopegp120Surface glycoprotein that binds CD4 TM - Transmembranegp41Transmembrane protein that anchors gp120 to virus, responsible for fusion between virus and host cell membrane.

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

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

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

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

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  B. T-cell activation (activates the production of MIP-1alpha and MIP-1beta in macrophages)

VIF: Virion Infectivity Factor

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

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