Retroviruses
1
Groups of Retroviruses
• Oncovirinae
important
Tumor viruses and similar
• Lentiviruses
important
Long latent period
Progressive chronic disease
Visna
HIV
• Spumavirinae
2
Retroviruses known to cause human cancer
• Human T cell lymphotropic virus -1 (HTLV-1)
Adult T cell leukemia, Sezary T-cell leukemia
Africa, Caribbean, Some Japanese Islands
• Human T cell lymphotropic virus -2 (HTLV-2)
Hairy cell leukemia
• HIV?
3
HIV and AIDS
Acquired Immunodeficiency Syndrome
• Disease caused by an infectious agent:
a retrovirus
4
HIV and AIDS
an infectious agent
In Los Angeles 1967-1978 only two cases of
Pneumocystis carinii pneumonia
• 1979 - 5 cases of Pneumocystis carinii
All Homosexual
With giemsa stain at
high magnification, the
faint bluish dot-like
intracystic bodies of
Pneumocystis carinii in
lung are seen in this
cytologic preparation
from a bronchoalveolar
lavage
5
HIV and AIDS
With dissemination to extrapulmonary sites, Pneumocystis carinii tends to
produce foci with prominent calcification, as seen in the kidney here grossly.
6
HIV and AIDS
an infectious agent – Kaposi’s Sarcoma
Early 1981 MMWR: 5 cases of
Kaposi’s sarcoma
Hitherto: rare (immunocompromization)
• 1981 - 26 cases of Kaposi’s sarcoma
• Young
• San Francisco and New York
• All Homosexuals
7
HIV and AIDS
Two rare diseases in the gay community linked to
IMMUNOSUPPRESSION
OPPORTUNISTIC INFECTIONS
Also
Lymphadenopathy
Hodgkin’s Lymphoma
• Gay-Related Immune Deficiency
• Acquired Immune Deficiency Syndrome (AIDS)
8
HIV and AIDS
Distinguishing characteristics
• Clusters of infected men
• Apparent concentration within sexually interactive groups
• High numbers of sex partners
Suggests an infectious agent
9
HIV and AIDS
More evidence for an infectious agent:
• Different ways of getting a similar syndrome
• Blood transfusions
• Intravenous drug use
• Hemophilia (clotting factor)
Female sex partners of AIDS-positive IV drug users and hemophiliacs
Not just in the Gay community
10
HIV and AIDS
Obvious agent:
A virus……that is now in the
blood supply
Primary route of transmission:
Sex
AIDS is a sexually-transmitted disease
11
HIV and AIDS
The Cellular Picture
Loss of one cell type throughout the course of the disease
CD4+ T4 helper cells
A fall in the CD4+ cells always precedes disease
In advanced disease the loss of another cell type
CD8+ cytotoxic killer cells
Suggests an infectious agent
A virus
12
AIDS Definition
• AIDS is currently defined in persons older than 13 years as the
presence of one of 25 conditions indicative of severe
immunosuppression
or
• HIV infection in an individual with a CD4+ cell count of <200 cells
per cubic mm of blood.
• AIDS is therefore the end point of an infection that is continuous,
progressive and pathogenic
• With the prevalence of HIV in the developing world, HIV and its
complications will be with us for generations
13
HIV and AIDS
The Virus
The virus only grows on T4 cells that are proliferating in response to an
immune stimulus
Therefore difficult to grow in culture
• Robert Gallo : reverse transcriptase in
activated T4 cells in blood of patients
with AIDS : HTLV-3
• Luc Montagnier: LAV
Human Immunodeficiency Virus (HIV)
14
15
GENOSOME
Diploid
Capped and polyadenylated
• Positive sense (same as mRNA)
Viral RNA cannot be read as mRNA
New mRNA must be made
Virus must make negative sense DNA before proteins are
made
Therefore virus must carry REVERSE TRANSCRIPTASE
into the cell
16
The Genome of HIV
•
Three structural genes
•
LTRs
•
Extra open reading frames are clue to latency
•
These ORFs code for small proteins - antibodies in AIDS patients
17
A HIV has:
3 structure genes
GAG : internal proteins
ENV: Envelope glycoproteins
POL: Enzymes
•Reverse transcriptase
•Integrase
•Protease (cuts polyproteins)
18
HIV - The Virus
Membrane: host derived
Two glycoproteins: gp160
gp120 and gp41
gp41 is fusogen that spans the membrane
sugars: immunosilent
vaccine problem
19
HIV - The Virus
Group-Specific Antigens
p17: inner surface
p24: nucleocapsid
p9: nucleocapsid associated with RNA
GAG gene
20
The Genome of HIV
Small non-structural proteins
mRNAs made by multiple splicing of genomic RNA
(c.f. mRNA for structural proteins)
EARLY
•TAT: TransActivator of Transcription
•REV: Regulator of Virion Protein Expression
•NEF: Negative Regulatory Factor
LATE
•VIF: Virion Infectivity Factor
TAT and REV are
essential for HIV
replication
•VPU: Viral Protein U
•VPR: Viral Protein R
21
HIV - Life History
A retrovirus
• Latency
• Specific destruction of CD4+ cells
•How does the virus enter the cell?
22
HIV - Life History
Entry into the cell
T4 (CD4+) cells are major target
Human HeLa
Cell
NOT INFECTED
Human HeLa Cell
transfected with CD4
antigen
INFECTED
But NOT the whole answer since this
does not happen if CD4 is transfected into a MOUSE cell
23
HIV - Life History
• Fusion at ambient pH
• No need for entry into
lysosomes
• Syncytia
Profound significance for AIDS
progression
Profound significance for therapy
24
HIV - Life History
Why do CD4-transfected human cells become infected
but CD4-transfected mouse cells do not?
Human cells must possess a co-factor for infection that mouse cells do not
Co-Receptors
CD8+ Cells
MIP-1 alpha MIP-1 beta RANTES
Chemokines
Block HIV infection of macrophages
25
HIV - Life History
HIV
chemokine
CD4
CD4
CCR5
CCR5
Mutant CCR5
CD4
macrophage
26
HIV and AIDS
Co-receptors
CCR5 is a chemokine receptor
25% of long term survivors are CCR5 or CCR2
mutants (deletions)
Many other chemokine receptors
27
HIV – life history
Endocytosis
Fusion of membranes
Release of nucleocapsid to cytoplasm
Nucleus
28
HIV - Life History
HIV carries with it:
Reverse transcriptase
• Integrase
• Protease
• tRNA primer
HIV genes
GAG POL ENV
HIV has no oncogene but could still be oncogenic
vaccine problem
29
HIV – life history
RNA-dependent DNA Polymerase encoded by virus
REVERSE TRANSCRIPTASE
RNA genome
Reverse transcriptase
virus
DNA genome
Integrase
virus
Integrates
Host RNA polymerase II
RNA genome
host
30
HIV – life history
Parental RNA
Reverse transcriptase
RNA/DNA Hybrid
Reverse
transcriptase
Linear DNA/DNA duplex
Circular Duplex DNA
Integrase
Host DNA
polymerase
Integration
Replication (DNA genome in cell)
Host RNA pol II
Transcription
Host splicing
enzymes
Viral RNA genome
mRNA
31
protein
RELEASE OF HIV
32
33
34
35
LIFE
HISTORY
OF
HIV
36
HIV - Life History
Latency - Cellular
Only activated T4 cells can replicate virus
Most infected T4 cells are rapidly lyzed but are replaced
Some T4 cells revert to resting state as memory cells which are long-lived
Memory T4 cells cannot replicate the virus unless the become activated
Macrophages do not show latency
Clinical Latency
HIV infection is not manifested as disease for years
During apparent clinical latency, virus is being replicated and cleared
37
Dynamics of CD4 T cells in an
HIV
infection
Cell death
Chronically-
apoptosis etc
Uninfected
infected memory
T cells with
provirus
Return to
resting
state
Infection
activated
T cell
Long lived!
Reactivation
Uninfected
unactivated
memory
T cell pool
Cell death
immune
destruction
Long lived!
Adapted from Saag and Kilby Nat
Med 5: 609, 1999
38
Latency
In the absence of any activating stimulus:
Homeostasis
39
Latency Breaks
Immune response
T4 resting
T4 activated
HIV production
40
HIV and AIDS
The cellular and immunological picture - The course of the disease
41
HIV and AIDS
The cellular and immunological picture - The course of the disease
42
HIV and AIDS
The cellular and immunological picture
The course of the disease
1. Acute Infection
• High virus titer
• Mild symptoms
• Fall in CD4+ cells but recovers
• Rise in CD8+ cells but recovers
• A high virus titer (up to 10 million viruses per ml blood)
• Macrophages infected
Macrophages bring HIV into the body if sexually transmitted
43
HIV and AIDS
2. A strong immune response
Virus almost disappears from circulation
• Good cytoxic T cell response
• Soluble antibodies appear later against both surface and
internal proteins
• Most virus at this stage comes from recently activated
(dividing) and infected CD4+ cells
• CD4+ cell production compensates for loss due to lysis
of cells by virus production and destruction of infected
cells by CTLs
44
HIV and AIDS
3. A latent state
Latency of virus and of symptoms
• Virus persists in extra-vascular tissues
• Lymph node dendritic cells
• Resting CD4+ memory cells (last a very
long time - a very stable population of cells)
carry provirus
45
HIV and AIDS
• 10 billion HIV particles per day
• Virus half life 5.7 hours
• 100-10 million virions per ml blood (set point)
• Small minority of T4 cells are infected
• Virus found in lymph nodes
46
HIV and AIDS
4. The beginning of disease
Massive loss of CD4+ cells
• CD4+ cells are the targets of the virus
• Cells that proliferate to respond to the
virus are killed by it: Clonal deletion
• Dendritic cells present antigen and virus
to CD4 cells just as they are activated
• Epitope variation allows more and more HIV to
escape from immune response just as response wanes
• Apoptosis of CD4+ cells
• HIV patients with high T4 cell counts
do not develop AIDS
47
HIV and AIDS
5. Advanced disease - AIDS
CD8+ cells destroy more CD4+ cells
• CD4 cell loss means virus and infected
cells no longer controlled
• As CD4+ cells fall below 200 per cu mm
virus titer rises rapidly and remaining
immune response collapses
• CD8+ cell number collapses
• Opportunistic infections
• Death in ~2 years without intervention
48
Inexorable decline of CD4+ T4
cells
Why do all of the
T4 cells
disappear?
At early stages of
infection only 1 in
10,000 cells is
infected
Late 1 in 40
Of great importance to therapeutic strategy
49
Virus destroys the cell as a result of
budding
But few cells are infected:
Early stage of infection 1:10,000
Late 1:40
HIV could kill sub population of precursor
cells
People develop AIDS even when they have
HIV that does not lyze cells
1. PUNCTURED
MEMBRANE
Why do all T4
cells
disappear?50
Why do all T4 cells disappear? 2
But syncytia not
common
Infected CD4 cell
Cells Fuse
Gp120 positive
Most T4 cells are
not HIV+
Could “sweep up”
uninfected cells
Killing of CD4 cells
2. Syncytium Formation
Uninfected CD4
cell
Gp120 negative
Syncytia may be
poor or ineffective
at immune
response
51
Why do all T4
cells
disappear?
Cytotoxic
T cell
Killing of CD4 cells
3. Cytotoxic T cell-mediated lysis
BUT: Most cells are
not infected
52
Killing of CD4+ cells
4. Binding of free Gp120 to
CD4 antigen makes
uninfected T4 cell look like
an infected cell
Complement-mediated lysis
Could account for the loss
of uninfected T4 cells
53
Why do all T4 cells disappear?
5. Apoptosis of T4 cells. Apoptosis of T4 cells is
normal in clonal deletion to overcome
autoimmunity
Also occurs with CD4 cells
54
CD8 cell
Macrophage
(no CD4 antigen)
gp120
HIV
chemokine
CXCR4
G protein
signal
?
?
Binding to CXCR4
results in
expression of TNFalpha receptor II
Binding to CXCR4
results in
expression of
TNF-alpha on the
cell surface
Apoptosis of T cells
55
CD8 cell
Macrophage
CXCR4
Death
CD8 T cell
apoptotic
bodies
56
HIV
Macrophages may be infected
by two routes
gp120
CD4
HIV gp120 binds to macrophage
CD4 antigen
Virus is opsonized by anti
gp120 antibodies which bind to
macrophage Fc receptors - an
enhancing antibody
Fc receptor
Anti-gp120
HIV
vaccine problem
57
Macrophages - The Trojan Horse
Early HIV isolated during infection are macrophage tropic (have a macrophage
chemokine co-receptor CCR5)
Virus probably infects patient via macrophages in semen
Infection by HIV leads to altered cytokine production
“slim disease”
Slim disease very like Visna in sheep - also infects macrophages
Macrophages form a reservoir outside the blood
Carry virus into different organs (brain)
Non-proliferating mature macrophages sustain
HIV production for a long time without being
killed by virus - - no latency
58
Population Polymorphism
HIV is a retrovirus
Retroviruses use host cell RNA polymerase II to replicate their genome
Pol II has a high error rate 1:2,000-10,000
HIV genome 9749 nucleotides
Therefore EVERY new virus has at least one mutation!
Every possible single mutation arises daily
1% of all possible double mutations arise daily
The HIV that infects a patient is very different from
that seen by the time AIDS appears
vaccine problem59
Population Polymorphism
• Initial infecting virus is macrophage-tropic (has CCR5 as
co-receptor)
• These are non-syncytium-inducing strains (INFECTIOUS)
(Note: most vaccines have been made against syncytiuminducing T4 cell tropic strains)
• As virus mutates, it changes subtypes of cells that it
infects as the ability to bind different co-receptors changes
60
Population Polymorphism
Early in infection:
• Macrophage-tropic
• Non-syncytium-inducing
• Slowly replicating
Late in infection
•T4 cell tropic
vaccine problem
• Syncytium-inducing
• High titer virus
61
Population Polymorphism
• The most variable protein is gp120
• Amino acid sequence within a single patient varies by 1-6%
• Up to 30% in population
vaccine problem
• Glycosylation masks conserved sites
vaccine problem
Co-infection may result in recombination
vaccine problem
62
Population Polymorphism
• Variation in reverse transcriptase leads to resistance to nucleoside
analogs
drug problem
• Variation in protease leads to resistance to protease inhibitors
drug problem
Polymorphism due to high mutation rate as a result of lack of
proof-reading in reverse transcriptase and RNA pol II
Sub-populations arise with altered cell tropism
63
Other cells infected by HIV
CD4• Epithelial cells of
bowel and vagina
• Endothelial cells of brain
• Brains cells : Astroglia, oligodendroglia
Galactocerebroside receptor
64
AIDS Statistics
• Approximately 40,000,000 people in the world are HIV-infected
• Approximately 8500 new HIV infections occur daily around the world
Over 90% of these are in developing countries.
1000 are in children less than 15 years of age.
Of adult infections, 48% are in women and 15% in individuals 15-25
years.
• As of June 2002, 793,026 Americans reported with AIDS. At least
457,667 of them have died. .
• Prior to the introduction of combination therapies for HIV, AIDS
65
incidence was increasing at a rate of just under 5% each year.
AIDS Statistics
Sub-Saharan Africa
• About 1 million new cases of AIDS per year
• 24 million people with HIV infection
• AIDS is responsible for a decrease in life expectancy and increase in child
mortality. Child mortality rates in East Africa will double by 2010 and adult life
expectancy has already declined by 2 years in that region.
• Several countries in sub-Saharan Africa report infection rates of 20-25%, especially
urban areas.
• Botswana: 35.8% of adult population infected
• In Zambia, 1 in 5 urban girls is HIV-positive by the age of 20
66
Anti-HIV Strategies
• Education
Sexually transmitted
Not highly infectious
• Chemotherapy
Mutation selection
Resistance
but
Suppress replication
No capacity for mutation
67
Anti-HIV Strategies
Highly
Active
AntiRetroviral
Therapy
HAART: Two nucleoside analog RT inhibitors and 1 protease inhibitor
Now also: Two nucleoside analog RT inhibitors and 1 non nucleoside68
Does HIV Cause AIDS?
Single common factor between:
• Gay San Franciscans
• New York I.V. drug users
• African heterosexuals
• Hemophiliacs
• Spouses of hemophiliacs and drug users
• Children of hemophiliacs and drug users
69
Does HIV Cause AIDS?
• HIV precedes AIDS in every population in which AIDS
occurs
• Infection by cloned virus
SIV
HIV
Simian AIDS
Human AIDS
70
Remember!
• Education led to leveling off
of rate of increase in AIDS
• HAART has greatly slowed death rate
•The fact that fewer people are dying per year from the
infection means that the number of HIV-infected people in
the population is rising!
• Unless education continues to be successful and unless
we can cure infected people of virus, the problem of virus
spread is and will continue to be with us
71