Download January 8, 2014 - HIV Structure, Lifecycle and Replication

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HIV Structure, Life Cycle, and Replication (1)
Dr. Matthew D. Marsden, Ph.D.
UCLA, Department of Medicine
[email protected]
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
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
(III)
Link
Virus-AIDS
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 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 HIVrelated 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)
Estimated
number of
Antiretroviral
people receiving
therapy coverage
antiretroviral
therapy
Estimated
number of
people needing
antiretroviral
therapy
10,600,000
Sub-Saharan Africa
37%
3,911,000
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
The Caribbean
51%
48%
425,000
52,400
840,000
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
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.
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/g
ene-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/
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/
Initial infection
Approximately 8-10 years
Asymptomatic period
(clinical latency)
Often (not always) accompanied by
severe flu like symptoms:
AIDS
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=9le
O28ydyfU
Viral RNA
gp120
p24
RT & other
virion
proteins
Fusion & Entry
Binding
Reverse
transcription
CD4
CXCR4
Nuclear
localization
& entry
Integration
Viral RNA
gp120
p24
Cellular Activation
Assembly
RT & other
virion prteins
Post-translational
processing
Budding
Translation
Viral Gene
Transcription
HIV Structure
Virion
Genomic
Proteomic
http://www.readcube.com/articles/10.1038/nrmicro2596
http://upload.wikimedia.org/wikipedia/commons/0/00/HI
V_Mature_and_Immature.PNG
HIV Structure
SIV
HIV
HIV Structure
Virion
Genomic
Proteomic
Referred to as:
• Provirus
• Proviral DNA
• Integrated Provirus
2 copies of viral RNA
gp120
p24
RT & other
virion
proteins
Fusion & Entry
Binding
Reverse
transcription
CD4
CXCR4
Nuclear
localization
& entry
Integration
HIV Genome
HIV RNA
R
U5
U3
R
Reverse transcription
HIV DNA
Integration
Host DNA
Host DNA
U3
R
LTR
U5
U3
R
LTR
U5
HIV-1 RNA
R
U5
U3
RRE
Nature Reviews Microbiology 2, 461-472 (June 2004)
R
Nature 460, 711-716 (6 August 2009)
HIV-1 Integrated DNA
Host DNA
Host DNA
U3
R
U5
LTR
U3
R
U5
LTR
CD4 & MHC
downregulation
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
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. 3.13
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
Structural protein expression and function
CD4 & MHC
downregulation
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/
Gene/Protein
gag
(Pr) 55gag
Mass, KDa
Function
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
myristate
Association with
membrane
Matrix p17
Envelope
Incorporation
Viral Entry
RNA binding
RNA packing
Formation of Gag
multimers
Capsid p24
p2
NC p9
p1
p6
Viral Budding
Source: BioAfrica Bioinformatics for HIV Research. http://bioafrica.mrc.ac.za/
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
transcriptase
p66/p51
p66 – copies RNA genome. RNAse H
p51 – regulatory?
Gag
p6
PR p10
RT
p51
p66
IN p32
Envelope
Source: The Molecular Biology of HIV/AIDS, D Ward, pp. 19
Gene/Protein
env
(Pr) 160 env
Mass, KDa
Function
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
gag
(Pr) 55gag
Mass, KDa
Function
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
transcriptase
p66/p51
p66 – copies RNA genome. RNAse H
env
(Pr) 160 env
p51 – functions in RT heterodimer
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
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
Positivefeedback
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
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)
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
Gene/Protein
tat
Tat
rev
Rev
Mass, KDa
Function
p14
Transactivates HIV transcription (kinase adaptor to RNAP2).
p19
Nuclear export of unspliced & single-spliced RNA (via RRE).
p27
Internalizes cell-surface CD4 & MHC Class I molecules.
nef
Nef
May enhance cellular activation.
vif
Vif
p23
Overcomes a post-entry block to infection.
May influence virion assembly.
vpu
Vpu
p16
Facilitates virion release via ion channel formation.
Targets CD4 for destruction in ER (freeing bound env).
vpr
Vpr
p15
Targets the HIV pre-integration complex to the nucleus.
Arrests activated cells in G 2 phase of cell cycle.
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