hiv structure, life cycle, and replication (1) dr. matthew d. marsden, ph.d. ucla, department of...

HIV Structure, Life Cycle, and Replication (1)

Dr. Matthew D. Marsden, Ph.D.UCLA, Department of Medicine

[email protected]

http://www.ck12.org/concept/HIV/

HIV Structure, Lifecycle and Replication (1)

Background: Basic Virology and Pathogenesis

Structure: Virion structure, genomic structure, and accessory molecules

Lifecycle: Infection and Expression

On http://aids.gov/hiv-aids-basics/hiv-aids-101/aids-timeline/

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.

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

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/

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

HIV is responsible for a catastrophic pandemic:

http://aids.gov

http://aids.gov

MSM = Men who have sex with menIDU = Intravenous drug users

ESTIMATES OF NEW HIV INFECTIONS IN THE U.S., 2009, BY TRANSMISSION CATEGORY

http://aidsvu.org/map/

http://aidsvu.org/map/

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.


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.

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?


Viruses are:


Can only replicate in living cells - Some can survive for long periods of time outside cells, but cannot replicate that way.

What is HIV?


Viruses are:


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.

http://tcf.epfl.ch/page-20833-en.html

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.

http://biology.kenyon.edu/slonc/gene-web/Lentiviral/Lentivi2.html

How does HIV cause disease?

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


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

Approximately 8-10 years

Initial infectionAsymptomatic period

(clinical latency) AIDSOften (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

http://www.youtube.com/watch?v=9leO28ydyfU

Movie of the HIV Life Cycle



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

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

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

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



HIV Structure

SIV HIV

HIV Structure

Virion

Genomic

Proteomic

CD4

CXCR4

Binding

Fusion & Entry

Nuclear localization & entry


Integration

Infection

gp120

p24

2 copies of viral RNA

RT & othervirion proteins

Referred to as:•Provirus•Proviral DNA•Integrated Provirus

R U5 RU3

HIV RNA

HIV DNA

R U5U3 R U5U3

LTR LTR

Host DNAHost DNA


Integration

HIV Genome

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

HIV-1 RNA

R U5 RU3

RRE

Nature 460, 711-716 (6 August 2009)

Modified from : The AmFAR AIDS Handbook, D Ward, pp. 348

HIV-1 Integrated DNA

R U5U3 R U5U3

LTR LTR

Host DNAHost DNA

CD4 & MHC downregulation

RNA Splicing

TranslationalFrameshift

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])

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

Initiation of HIV transcription is performed by cellular factors

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

CD4 & MHC downregulation

Structural protein expression and function


Gag and Gag-Pol

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

Gag and Gag-Pol

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

Matrix p17 Capsid p24 NC p9 p6p1p2

myristate

Association withmembrane

Formation of Gagmultimers

RNA bindingRNA packing

Viral Entry

EnvelopeIncorporation

Viral Budding

NC - Nucleocapsid p7/p9

Precise location in virion unknown, not generally present inother retroviruses, may aid in incorporation of Vpr into virion

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

p6 p6

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?



Envelope

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 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 p7/p9

Precise location in virion unknown, not generally present inother retroviruses, may aid in incorporation of Vpr into virion

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 – functions in RT heterodimerenv

(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

p6 p6


Regulatory proteins

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

TAT: Trans-Activator of Transcription

TAR

Poor transcription

Recruitment ofCDK9

Enhanced transcription

CDK9

Tat

Tat

Tat

TatTat

TAT: Trans-Activator of Transcription

Positive-feedback loop

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

* 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• Counteracts Tetherin (Bst2/CD317/HM1.24)

• Downregulation of CD4

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003299

Tetherin

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003299

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 .

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

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

hiv structure, life cycle, and replication (1) dr. matthew d. marsden, ph.d. ucla, department of...

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