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

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 ImmunodeficiencyMichael 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

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 AIDSGallo 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 epidemicTuofu Zhu, Bette T. Korber, Andre J. Nahmiask,Edward Hooper, Paul M. Sharp & David D. Ho. February 1998Nature, Vol 391, 5

NEW HOMOSEXUAL DISORDER WORRIES HEALTH OFFICIALSBy LAWRENCE K. ALTMAN. Published: May 11, 1982

From GRID to AIDS

The emergence of HIV/AIDS in the Americas and beyondM. Thomas P. Gilbert, Andrew Rambaut, Gabriela Wlasiuk, Thomas J. Spira, Arthur E. Pitchenik, and Michael WorobeyPNAS , November 20, 2007, 18566–18570

Pneumocystis carinii Pneumonia and Mucosal Candidiasis in Previously Healthy Homosexual Men — Evidence of a New Acquired Cellular ImmunodeficiencyMichael 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

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/

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

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 ChimpanzeeHIV-2 Lower Low West Africa Sooty Mangabey

CRF14_BG

HIV phylogeny

www.hivmedicine.com/textbook/pathogen.htm

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 & othervirion proteins

Expression

gp120

p24

Viral RNA

RT & othervirion prteins

Budding

Assembly

Viral Gene Transcription

Translation

Post-translationalprocessing

Cellular Activation

HIV Structure

Virion

Genomic

Proteomic

HIV Structure

SIV HIV

HIV Structure

Virion

Genomic

Proteomic

CD4

CXCR4

Binding

Fusion & Entry

Nuclear localization & entry

Reverse transcription

Integration

Infection

gp120

p24

Viral RNA

RT & othervirion proteins

R U5 RU3

HIV RNA

HIV DNA

R U5U3 R U5U3

LTR LTR

Host DNAHost DNA

Reverse transcription

Integration

HIV Genome

Nature Reviews Microbiology 2, 461-472 (June 2004)

Nature 460, 711-716 (6 August 2009)

HIV-1 RNA

R U5 RU3

Source: The AmFAR AIDS Handbook, D Ward, pp. 348

HIV-1 Integrated DNA

R U5U3 R U5U3

LTR LTR

Host DNAHost DNA

RNA Splicing

TranslationalFrameshift

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. http://www.123bio.net/

Source: Atlas of Infectious Diseases, Mandell &

Mildvan (ed.), pp. 3.13

HIV Structure

Virion

Genomic

Proteomic

HIV Proteins

Structural ProteinsGag: Matrix, Capsid, NC, p6 Pol: Protease, Reverse Transcriptase, Integrase Env: gp120, gp41

Regulatory ProteinsTatRevNef

Accessory proteinsVifVprVpu

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

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

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 inother retroviruses, may direct proteins to secretory path of cell.

p9 – Assoc. with HIV RNA, involved in packaging of HIVRNA into virions

Matrix p17 Capsid p24 NC p9 p6p1p2

myristate

Association withmembrane

Formation of Gagmultimers

RNA bindingRNA packing

Viral Entry

EnvelopeIncorporation

Viral Budding

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

PR p10 RT IN p32p6

Gag

p51

p66

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 – reversetranscriptase

p66/p51 p66 – copies RNA genome. RNAse H

p51 – regulatory?

Gene/Protein Mass, KDa Function

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 InhibitorsIsentress (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 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

Gene/Protein Mass, KDa Functiongag

(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 inother retroviruses, may direct proteins to secretory path of cell.

p9 – Assoc. with HIV RNA, involved in packaging of HIVRNA 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 – reversetranscriptase

p66/p51 p66 – copies RNA genome. RNAse H

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.

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

Regulatory Proteins:TAT: Trans-Activator of TranscriptionREV: Regulator of Virion protein expressionNEF: Negative Regulatory Factor

Accessory Proteins:VIF: Virion Infectivity FactorVPU: Viral Protein UVPR: Viral Protein R

TAR

Poor transcription

Recruitment ofCDK9

Enhanced transcription

CDK9

Tat

Tat

Tat

TatTat

Release to extracellular medium:Apoptosis bystander T-cellsCXCR4 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

www.hivmedicine.com/textbook/pathogen.htm

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

G ene/Pro te in M ass , K D a Functionta t

Tat p14 Transactiva tes H IV transcrip tion (k inase adaptor to R N A P 2).

revR ev p19 N uclear export o f unsp liced & s ing le -sp liced R N A (v ia R R E ).

nef

N ef p27 In terna lizes ce ll-surface C D 4 & M H C C lass I m o lecu les .

M ay enhance ce llu la r ac tiva tion .

v if

Vif p23 O vercom es a post-en try b lock to in fec tion .

M ay in fluence v irion assem bly .

vpu

Vpu p16 Fac ilita tes v irion re lease v ia ion channe l fo rm ation .

Targets C D 4 fo r destruction in ER (free ing bound env).

vprVpr p15 Targets the H IV p re -in tegra tion com plex to the nuc leus .

A rres ts activa ted ce lls in G 2 phase o f ce ll cyc le .

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