Transcript HIV Attachment & Entry: Insights into pathogenesis and

HIV
Cellular Pathogenesis I
Benhur Lee, M.D.
Adults and children estimated to be living
with HIV/AIDS as of end 2001
North America
940 000
Caribbean
420 000
Latin America
1.4
million
Eastern Europe
& Central Asia
Western Europe
560 000 1 million
North Africa
& MiddleEast
440 000
Sub-Saharan
Africa
East Asia & Pacific
1 million
South
& South-East Asia
6.1 million
28.1
million
Total: 40 million
Australia
& New
Zealand
15 000
Estimated number of adults and children
newly infected with HIV during 2001
North America
45 000
Caribbean
60 000
Latin America
130 000
Eastern Europe
Western Europe
30 000 & Central Asia
250 000
North Africa
& Middle East
80 000
Sub-Saharan
Africa
East Asia & Pacific
270 000
South
& South-East Asia
800 000
3.4 million
Australia
& New
Zealand
500
Total: 5 million
Estimated adult and child deaths
from HIV/AIDS during 2001
North America
20 000
Caribbean
30 000
Latin America
80 000
Eastern Europe &
Central Asia
Western Europe23 000
6 800
East Asia & Pacific
North Africa
& Middle East
30 000
Sub-Saharan
Africa
35 000
South
& South-East Asia
400 000
2.3 million
Australia
& New
Zealand
120
Total: 3 million
See Quicktime Movie file
HIV: 15 proteins (9 genes) and an RNA
~10 kb
Pox viruses: >100 ORFs
(e.g. Smallpox)
~200 kb
Lipid
Membrane
Derived
From
Host Cell
Corecept or Binding Sit e
Exposed- -HighlyConserv ed
gp41
Corecept or Binding:
target f or small molecule
inhibitors
gp41
C D4
gp12 0
V1
V2
V3
gp12 0
V3
gp12 0
V2
V1
gp41
gp12 0
V1/ V2
gp41
gp12 0
V3
Coreceptor
CD4 Binding
Expos ur e of
Cor e ce ptor
Binding Site
Cor e ce ptor
Binding
Fus ion Pe ptide
Ins e r tion
Hair pin For m ation
---> m e m brane fus ion
Virus Membrane
Fusion
No Fusion
Fusion
peptide
Cell membrane
T-20
&
Reverse Transcription
Productive uncoating requires a cellular
chaperone protein (Cyclophilin A)
•
Chaperones
– facilitate protein folding and
assembly
– has peptidylprolyl isomerase
activity
•
Viruses made without cyclophilin
A cannot undergo reverse
transcription but are fusion
competent
– Point mutations in capsid
that cannot bind cyclophilin
A
– Viruses produced in the
presence of Cyclosporine A
•
Efficient RT may require
expansion or partial disassembly
of the viral capsid core
(-)Strong-stop
DNA
RT
(+)Strong-stop
DNA
Pre-Integration Complex
Integration
1)Processing (Integrase)
2)Joining (Integrase)
3)Repair (Host cell)
Reverse Transcription Products: Early
R
U5
Late
Cytoplasm
Nucleus
LTR
Transcription
(tat)
rev-mediated
Requires protease (PR)
to process structural
proteins (Gag)
Protease Inhibitors
prevent viral assembly
(used in combination
with RT-Inhibitors
in HAART)
Requires human/primate
co-factors: Gag processing
is deficient in murine cells
Assembly and budding
takes place at lipid rafts:
cholesterol-rich membrane
microdomains
3. Trigger Event
pH-independent
receptor dependent
HIV
4. Conformational Change
5. Membrane Fusion
Enveloped Virus
Entry
H+
H+
1. Attachment
2. Receptor
Engagement
H+
H+
H+
H+
Influenza
Endosome
3. Trigger Event
pH-dependent
Attachment but not entry
CD4
CD4 is necessary but
not sufficient for viral
entry
CD4 mediates viral
attachment to cell surface
but not membrane fusion
Mouse Cell
Attachment & Entry
?
Cofactor/Coreceptor
required
Human Cell
CD4
(Early)
(Late)
M-tropic
T-tropic
Coreceptor A
Coreceptor B
Coreceptor B
Coreceptor A
CD4+
CD4+
CD4+
Primary T cells
Macrophages
T cell Line
CAF
(Cellular
Antiviral Factor)
HIV-1
?
No Viral
Replication
Supernatant
CD8+ T-cells
CD4+ T-cells
RANTES (CCL5)
MIP-1a (CCL3)
MIP-1b (CCL4)
Viral Inhibition Activity
Chemokines
HPLC Fractionation
45
1
Fraction #
Supernatant
Biochemical
Confirmation
WB, IP, and
Neutralization
Expt.
Purify,
Mass Spec or
Protein sequencing
CD8+ T-cells
Database searching
Chemokines
--chemotactic cytokines
--8-10 kDa
--binds to 7-TM GPCR
--classified based on number and
spacing of conserved cysteine
residues
--CC, CXC, CX3C, XC
CAF
(Cellular
Antiviral Factor)
“T-tropic” HIV
Expression Cloning
Identifies a Chemokine
Receptor as Coreceptor for
T-tropic Viral Entry
CD4
Fusin (CXCR4)
vacEnv
vacT7-Pol
Cell-Cell Fusion
Effector Cell
HIV Env
(gp160)
T7
T7-Pol
CD4
Target Cell
vacT7-b-gal
T7
b-gal
Transfect
pools of
cDNA library
b-gal
Expression Cloning Identifies a Chemokine
Receptor as Coreceptor for T-tropic Viral Entry
“T-tropic” HIV
1000 cDNA
pools
(@ 4x103 each)
CD4
1000 cDNA
pools
(@ 4 each)
Screen
Plasmids from
Individual
Colonies
Fusin (CXCR4)
Sequence positive plasmid
Confirmation of Coreceptor
Function of Fusin (CXCR4)
• Fusion induced only when expressed with CD4
• Fusion not induced with uncleaved (nonfunctional) Env
• Fusin (CXCR4) expression confers cell fusionpromoting activities in a variety of CD4expressing cell lines (that were otherwise nonpermissive)
• Endogenous expression of Fusin in a variety of
permissive cell lines
• Antibody to Fusin inhibits cell fusion
CCR5 binds
RANTES,
MIP-1a &
MIP-1b
Use of Different Coreceptors Largely
Accounts for Viral Tropism
T-tropic
M-tropic
CD4+
CD4+
CD4+
CCR5+ CXCR4+
Primary T cells
CCR5+
CXCR4+
Macrophages
T cell Line
ccr5/ccr5
81% of Caucasian
Get infected normally
Progress to AIDS normally
ccr5/Dccr5
15-18% of Caucasians
Get infected normally
But progress to AIDS 2-4 years
more slowly
Dccr5 /Dccr5
1% of Caucasians
Highly Resistant to Infection
Loss of CCR5 function but
otherwise normal (no side effects)
This makes CCR5 an attractive drug target
Polymorphism in 3’ untranslated
region of SDF-1 gene may be
Chemokine Ligand
associated with delayed
progression to AIDS
SDF-1
SDF-1[3’A/3’A]
SDF-1[+/3’A]
SDF-1[+/+]
CXCR4
CXCR4/SDF-1 Knock-Out Mouse
phenotype is lethal
CCR5 and CXCR4 are the major
Coreceptors for HIV-1/HIV-2 entry
• All HIV-1/HIV-2/SIV strains
use either CCR5, CXCR4 or
both
• SIV can efficiently use
alternate coreceptors:
GPR15(BOB),
CXCR6(Bonzo/STRL33),
GPR1
• Polymorphisms in
chemokines or their receptors
have been reported to
modulate the course of HIV
disease.
• Other Chemokine receptors
with Coreceptor activity (in
vitro)
–
–
–
–
–
–
CCR2
CCR3
CCR8
CX3CR1
APJ
US28 (from CMV)
Experimental Strategies
for Identification of
Viral Cellular Receptors
Impact of Coreceptor Discoveries On:
• Virus Entry Mechanism: structure-function, how does
Env interact with coreceptor, can fusion be inhibited?
•
(Lee et al., Science 281:487; Lee et al., J. Biol. Chem. 274:9617; Blanpain and Lee et al, J. Biol.
Chem., 274: 18902; Wang and Lee et al., J. Biol. Chem. 274: 28413, Blanpain et al., Blood, 94:
1899)
• Pathogenesis:does differential utilization (expression) of
coreceptors account for the variable outcomes and
pathological syndromes associated with HIV infection
•
(Lee et al, Blood 93: 1145; Hariharan et al, Blood 93: 1145; Lee et al., Proc Natl Acad Sci USA 96:
5215; Honczarenko et al., Blood 94: 2990, Majka et al., J. Clin. Invest. 104: 1739, Lee et al., Eur.J.
Immunol., in press)
• Coreceptor Polymorphisms: consequences for disease
progression (CCR2-V64I, CCR5 promoter)
•
(Lee at al., J. Virol.72: 7450; Martin et al., Science 282: 907)
gp120
CD4
Mutations affecting gp120
Binding to CCR5
>90% >75% >50%
Degree of Conservation
Most
Binding
Least
Virus Membrane
Cell Membrane
Corecept or Binding Sit e
Exposed- -HighlyConserv ed
gp41
Corecept or Binding:
target f or small molecule
inhibitors
gp41
C D4
gp12 0
V1
V2
V3
gp12 0
V3
gp12 0
V2
V1
gp41
gp12 0
V1/ V2
gp41
gp12 0
V3
Coreceptor
CD4 Binding
Expos ur e of
Cor e ce ptor
Binding Site
Cor e ce ptor
Binding
Fus ion Pe ptide
Ins e r tion
Hair pin For m ation
---> m e m brane fus ion
Virus Membrane
Fusion
Small Molecular Antagonists
1)TAK779
2)Sch-C
3)E914
Modified Chemokines
1)AOP-RANTES
Monoclonal Abs
No Fusion
Fusion
peptide
Cell membrane
T-20
Entry of HIV is triggered by receptor
engagement
Viral Attachment Factors
DC-SIGN (CD209)
• C-type (calcium-dependent) lectin
• Relatively specific expression on subsets of
dendritic cells in tissue
• Binds to HIV Env (gp120) with greater
affinity than CD4 (Kd 1.3nM vs 4.5 nM)
• Binding to gp120 can be competed off with
certain sugars (mannan>L-fucose>amethyl-D-mannoside)
DC-SIGN: A Conduit for Transfer of
HIV to Lymphoid Organs?
Geijtenbeek, T.B.H. et al, Cell 100: 594
DC-SIGN mediates HIV-1 infection
in DC-T-cell co-culture
DC-SIGN enhances HIV
infection in trans
Consequences of DC-SIGN Mediated High
Affinity Viral Attachment
• Altered or expanded viral tropism due to
infection of target cells with
– Lower CD4 levels
– Lower coreceptor levels
– Alternate coreceptors (e.g. Bonzo/STRL33)
Some DC-SIGN transduced T-cell lines can
support R5-Viral Replication
Jurkat
SupT1
Jurkat and SupT1 cells do not express detectable levels
of CCR5 by FACS
R5 Viral Infection of Jurkat/DC-SIGN+
cells are CD4 and CCR5 dependent
(CCR5 Antagonist)