HIV Structure, Life Cycle, and Replication (1) http://www.ck12.org/concept/HIV/ HIV Structure, Life Cycle, and Replication (1) Dr. Matthew D. Marsden, Ph.D. UCLA, Department of Medicine mmarsden@ucla.edu

HIV Structure, Lifecycle and Replication (1) Background: Basic Virology and Pathogenesis Structure: Virion structure, genomic structure, and accessory molecules Lifecycle: Infection and Expression

A new disease… On http://aids.gov/hiv-aids-basics/hiv-aids-101/aids-timeline/

A new disease… By the end of 1981, there was a cumulative total of 270 reported cases of severe immune deficiency among gay men, and 121 of those individuals had died. In 1983, Luc Montagnier and Françoise Barré-Sinoussi reported the discovery of a new virus (later called HIV) that is the cause of AIDS. The first commercial blood test for HIV was licensed in 1985, allowing screening of the U.S. blood supply. In 1987 the first anti-HIV drug (AZT) was approved by the U.S. Food and Drug Administration. The first potent combination of anti-HIV drugs became available in 1995. On http://aids.gov/hiv-aids-basics/hiv-aids-101/aids-timeline/

(I) Identification of AIDS (II) Isolation of the virus (III) Link 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 (III) Link Virus-AIDS 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

Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). Since the epidemic was identified 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/ Duesberg hypothesis $19,912 a year for a single patient (2010) http://www.unaids.org/en/

Clinton Health Access Initiative (CHAI) 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 Africa 37% 3,911,000 10,600,000   Eastern and Southern Africa 41% 3,203,000 7,700,000 Western and Central Africa 25% 709,000 2,900,000 Latin America and the Caribbean 50% 478,000 950,000 Latin America 51% 425,000 840,000 The Caribbean 48% 52,400 110,000 East, South and South-East Asia 31% 739,000 2,400,000 Europe and Central Asia 19% 114,000 610,000 North Africa and the Middle East 11% 12,000 100,000 Total 36% 5,254,000 14,600,000 Clinton Health Access Initiative (CHAI) $25 billion total needed to achieve all targets in low and middle-income countries (2009) On average it cost $380 to keep a patient on the medication for a year in Global Fund countries Clinton Health Access Initiative (CHAI) $25 billion total needed to achieve all targets in low and middle-income countries (2009) On average it cost $380 to keep a patient on the medication for a year in Global Fund countries

HIV is responsible for a catastrophic pandemic:

http://aids.gov

http://aids.gov MSM = Men who have sex with men ESTIMATES OF NEW HIV INFECTIONS IN THE U.S., 2009, BY TRANSMISSION CATEGORY MSM = Men who have sex with men IDU = Intravenous drug users

http://aidsvu.org/map/

http://aidsvu.org/map/

What is HIV?

What is HIV? To understand what HIV and AIDS are, let’s break it down: Causative agent: H – Human – This particular virus can only infect human beings. I – Immunodeficiency – HIV weakens your immune system by destroying important cells that fight disease and infection. A "deficient" immune system can't protect you. V – Virus – A virus can only reproduce itself by taking over a cell in the body of its host. http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/

What is HIV? To understand what HIV and AIDS are, let’s break it down: Causative agent: H – Human – This particular virus can only infect human beings. I – Immunodeficiency – HIV weakens your immune system by destroying important cells that fight disease and infection. A "deficient" immune system can't protect you. V – Virus – A virus can only reproduce itself by taking over a cell in the body of its host. Disease: A – Acquired – AIDS is not something you inherit from your parents. You acquire AIDS after birth. I – Immuno – Your body's immune system includes all the organs and cells that work to fight off infection or disease. D – Deficiency – You get AIDS when your immune system is "deficient," or isn't working the way it should. S – Syndrome – A syndrome is a collection of symptoms and signs of disease. AIDS is a syndrome, rather than a single disease. It is a complex illness with a wide range of symptoms. http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/

What is HIV? Human immunodeficiency virus or HIV is a type of virus (from the Latin “virus” referring to poison). Viruses are: Small Generally too small to see with a regular light microscope (20 - 300 nm diameter) If a cell was a football stadium then a small virus would be around the size of a football.

What is HIV? Human immunodeficiency virus or HIV is a type of virus (from the Latin “virus” referring to poison). Viruses are: Small Generally too small to see with a regular light microscope (20 - 300 nm diameter) If a cell was a football stadium then a small virus would be around the size of a football. Can only replicate in living cells - Some can survive for long periods of time outside cells, but cannot replicate that way.

What is HIV? Human immunodeficiency virus or HIV is a type of virus (from the Latin “virus” referring to poison). Viruses are: Small Generally too small to see with a regular light microscope (20 - 300 nm diameter) If a cell was a football stadium then a small virus would be around the size of a football. Can only replicate in living cells - Some can survive for long periods of time outside cells, but cannot replicate that way. Made up of Nucleic acids (DNA/RNA) and proteins -different from protein-only “prions” or nucleic acid-only “viroids”. Thousands of very different types of virus exist and HIV is a particular type termed a “retrovirus”.

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 of reproducing 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. Phage, Ebola, Rabies, Herpes, HIV

The HIV genome is composed of 9 genes, which encode 15 proteins. http://tcf.epfl.ch/page-20833-en.html http://biology.kenyon.edu/slonc/gene-web/Lentiviral/Lentivi2.html For comparison, the E. Coli bacterium contains around 4,377 genes and the human genome encodes around 21,000 genes.

How does HIV cause disease?

HIV is a virus that infects and destroys cells of the immune system (CD4+ cells). http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/

Approximately 8-10 years Asymptomatic period (clinical latency) HIV is a virus that infects and destroys cells of the immune system (CD4+ cells). http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/ Approximately 8-10 years Asymptomatic period (clinical latency) Initial infection AIDS Often (not always) accompanied by severe flu like symptoms: Opportunistic infections and cancer: AIDS (acquired immunodeficiency syndrome) is the late-stage HIV disease. This occurs when immune system becomes so damaged that it cannot fight off diseases and certain types of cancer.

HIV Life cycle http://preprod.www.tibotec.com/content/backgrounders/www.tibotec.com/hiv_lifecycle.html

Movie of the HIV Life Cycle http://www.youtube.com/watch?v=9leO28ydyfU

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

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

HIV Structure Virion Genomic Proteomic

http://www.readcube.com/articles/10.1038/nrmicro2596

http://upload. wikimedia http://upload.wikimedia.org/wikipedia/commons/0/00/HIV_Mature_and_Immature.PNG

HIV Structure SIV HIV Nature 441, 847-852 (15 June 2006) Distribution and three-dimensional structure of AIDS virus envelope spikes 10 spikes per virus

HIV Structure Virion Genomic Proteomic

Infection Referred to as: 2 copies of viral RNA Provirus gp120 Proviral DNA Integrated Provirus 2 copies of viral RNA gp120 p24 RT & other virion proteins Infection Fusion & Entry Intro to the HIV cycle Binding Reverse transcription Nuclear localization & entry Integration CD4 CXCR4

HIV Genome HIV RNA HIV DNA LTR Reverse transcription Integration R U5 Host DNA

HIV-1 RNA R U5 U3 Nature Reviews Microbiology 2, 461-472 (June 2004) RRE IRES: Internal Ribosomal Entry Site DIS: Dimerization loop SD: Splicing donor TAR: Transactivation Response element for tat

Nature 460, 711-716 (6 August 2009)

Modified from : The AmFAR AIDS Handbook, D Ward, pp. 348 HIV-1 Integrated DNA R U5 U3 LTR Host DNA CD4 & MHC downregulation 9,749 nucleotides. Nine genes: gag, pol, vif, vpr, tat, rev, vpu, env, nef 15 proteins: matrix, capsid, p6; NC (p7), protease, reverse transcriptase, integrase, vif, vpr, tat, rev, vpu, gp120, gp41, nef Modified from : The AmFAR AIDS Handbook, D Ward, pp. 348

RNA Splicing Translational Frameshift

Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19 Slow mRNA production: Tat Unstable mRNA: Rev Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19 These are the major spliced RNA species, but HIV can actually produce at least 109 different spliced RNAs (Nucleic Acids Res. 2012 Nov 1;40[20])

Initiation of HIV transcription is performed by cellular factors Source: Atlas of Infectious Diseases, Mandell & Mildvan (ed.), pp. 3.13

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

Source: Atlas of Infectious Diseases, Mandell & Mildvan (ed. ), pp. 3

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 10 spikes, 1,500 gag molecules, 100 gag-pol

Structural protein expression and function CD4 & MHC downregulation

Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19 Gag and Gag-Pol Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19

Gag and Gag-Pol Source: BioAfrica Bioinformatics for HIV Research. http://bioafrica.mrc.ac.za/

Aids nuclear import and viral assembly 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 p6 p6 Precise location in virion unknown, not generally present in other retroviruses, may aid in incorporation of Vpr into virion NC - Nucleocapsid p7/p9 Assoc. with HIV RNA, involved in packaging of HIV RNA into virions Association with membrane Formation of Gag multimers RNA binding RNA packing myristate Matrix p17 Capsid p24 p2 NC p9 p1 p6 Envelope Incorporation Viral Budding Viral Entry

Source: BioAfrica Bioinformatics for HIV Research. http://bioafrica

Cleaves Gag and Gag-Pol 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 p6 PR p10 RT IN p32 p51 p66

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

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.

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 p6 p6 Precise location in virion unknown, not generally present in other retroviruses, may aid in incorporation of Vpr into virion NC - Nucleocapsid p7/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 – functions in RT heterodimer env (Pr) 160 env gp160 Env precursor protein SU – Envelope gp120 Surface glycoprotein that binds CD4 and coreceptors TM - Transmembrane gp41 Transmembrane 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 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

TAT: Trans-Activator of Transcription TAR Poor transcription

TAT: Trans-Activator of Transcription TAR Recruitment of CDK9 Positive- feedback loop Poor transcription Tat Tat Tat Tat Tat CDK9 Enhanced transcription

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

NEF: Negative Regulatory Factor * Downmodulation the expression of several surface molecules important in host immune function: MHC I, MHC II, CD4 * T-cell activation (activates the production of MIP-1alpha and MIP-1beta in macrophages)

VIF: Virion Infectivity Factor Nature Reviews Immunology 4, 868-877 (November 2004) Retroviral restriction by APOBEC proteins www.hivmedicine.com/textbook/pathogen.htm

Involved in viral budding, enhancing virion release from the cell VPU: Viral Protein U Involved in viral budding, enhancing virion release from the cell Counteracts Tetherin (Bst2/CD317/HM1.24) Tetherin http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003299 Downregulation of CD4

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

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 at least 2 different classes in combination allows potent inhibition of HIV replication.

Take-home points: Structure: Env is only exposed viral protein in virion (neutralization resistant) Binding/fusion with host cell is 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 HIV protease activity is needed for generation of infectious virions accessory genes modulate: 1) cellular function (e.g., nef, vpr) 2) viral gene expression (e.g., tat, rev) Capsid (p24) represents the primary structural component of virion small molecule inhibitors of these structure’s functional activity represent the primary current antiviral therapeutic strategies