1. HIV Structure, Life Cycle, and Replication (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. A new disease… On

4. 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

5. (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:
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
(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 , pp
(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

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.
Duesberg hypothesis
$19,912 a year for a single patient (2010)

8. 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

9. HIV is responsible for a catastrophic pandemic:

11. 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

14. What is HIV?

15. 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.

16. 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.

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.
Phage, Ebola, Rabies, Herpes, HIV

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.

22. How does HIV cause disease?

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

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

25. HIV Life cycle

26. Movie of the HIV Life Cycle

27. 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

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

29. HIV Structure
Virion
Genomic
Proteomic

31. http://upload. wikimedia

32. 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

35. HIV Structure
Virion
Genomic
Proteomic

36. 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

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

38. 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

39. Nature 460, (6 August 2009)

40. 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

41. RNA Splicing
Translational
Frameshift

42. 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 Nov 1;40[20])

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

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

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

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

48. Structural protein expression and function
CD4 & MHC downregulation

49. 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

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

51. 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

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

53. 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

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

55. 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.

56. 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

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

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. TAT: Trans-Activator of Transcription
TAR
Poor transcription

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

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

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
Nature Reviews Immunology 4, (November 2004)
Retroviral restriction by APOBEC proteins

64. 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
Downregulation of CD4

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

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