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HIV Structure, Life Cycle, and Replication (1) Dr. Matthew D. Marsden, Ph.D. UCLA, Department of Medicine

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Presentation on theme: "HIV Structure, Life Cycle, and Replication (1) Dr. Matthew D. Marsden, Ph.D. UCLA, Department of Medicine"— Presentation transcript:

1 HIV Structure, Life Cycle, and Replication (1) Dr. Matthew D. Marsden, Ph.D. UCLA, Department of Medicine

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

3 On A new disease…

4 On 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.

5 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

6 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 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.

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8 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 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

9 HIV is responsible for a catastrophic pandemic:

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

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14 What is HIV?

15 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. What is HIV?

16 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. What is HIV? 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.

17 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 ( nm diameter) If a cell was a football stadium then a small virus would be around the size of a football.

18 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 ( 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.

19 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 ( 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”.

20 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.

21 The HIV genome is composed of 9 genes, which encode 15 proteins. For comparison, the E. Coli bacterium contains around 4,377 genes and the human genome encodes around 21,000 genes. ene-web/Lentiviral/Lentivi2.html

22 How does HIV cause disease?

23 HIV is a virus that infects and destroys cells of the immune system (CD4+ cells).

24 HIV is a virus that infects and destroys cells of the immune system (CD4+ cells). Approximately 8-10 years Initial infection Asymptomatic period (clinical latency) 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.

25 HIV Life cycle

26 O28ydyfU Movie of the HIV Life Cycle

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

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

29 HIV Structure Virion Genomic Proteomic

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31 V_Mature_and_Immature.PNG

32 HIV Structure SIVHIV

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35 HIV Structure Virion Genomic Proteomic

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

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

38 Nature Reviews Microbiology 2, (June 2004) HIV-1 RNA RU5RU3 RRE

39 Nature 460, (6 August 2009)

40 Modified from : The AmFAR AIDS Handbook, D Ward, pp. 348 HIV-1 Integrated DNA RU5 U3 RU5 U3 LTR Host DNA CD4 & MHC downregulation

41 RNA Splicing Translational Frameshift

42 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 Nov 1;40[20])

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

44 Dr. Isabelle BOUALLAGA Institut Pasteur.

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

46 HIV Structure Virion Genomic Proteomic

47 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

48 CD4 & MHC downregulation Structural protein expression and function

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

50 Source: BioAfrica Bioinformatics for HIV Research. Gag and Gag-Pol

51 Gene/ProteinMass,KDaFunction gag (Pr) 55gagp55Gag precursor protein MA - Matrixp17Aids nuclear import and viral assembly CA - Capsidp24HIV central core – contains HIV genome and enzymes Matrix p17Capsid p24NC p9p6p1p2 myristate Association with membrane Formation of Gag multimers RNA binding RNA packing Viral Entry Envelope Incorporation Viral Budding NC - Nucleocapsidp7/p9 Precise location in virion unknown, not generally present in other retroviruses, may aid in incorporation of Vpr into virion Assoc. with HIV RNA, involved in packaging of HIV RNA into virions p6

52 Source: BioAfrica Bioinformatics for HIV Research.

53 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

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

55 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

56 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 - Nucleocapsidp7/p9 Precise location in virion unknown, not generally present in other retroviruses, may aid in incorporation of Vpr into virion 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 – functions in RT heterodimer env (Pr) 160 envgp160Env precursor protein SU – Envelopegp120Surface glycoprotein that binds CD4 and coreceptors TM - Transmembranegp41Transmembrane protein that anchors gp120 to virus, responsible for fusion between virus and host cell membrane p6

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

58 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

59 TAR Poor transcription TAT: Trans-Activator of Transcription

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

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

62 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)

63 VIF: Virion Infectivity Factor

64 VPU: Viral Protein U Involved in viral budding, enhancing virion release from the cell Counteracts Tetherin (Bst2/CD317/HM1.24) Downregulation of CD4 Tetherin

65 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

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67 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.

68 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


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