the role of pi3k signaling pathways in hiv-1 infection of resting cd4+t-cells
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The role of PI3K signaling pathways in HIV-1 infection of resting CD4+T-cells. Suha Saleh, Paul Cameron, Georgina Sallmann, Anthony Jaworowski, and Sharon Lewin Monash University, Melbourne, Australia. Background:. - PowerPoint PPT PresentationTRANSCRIPT
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The role of PI3K signaling pathways in HIV-1 infection of resting
CD4+T-cellsSuha Saleh, Paul Cameron, Georgina Sallmann,
Anthony Jaworowski, and Sharon Lewin
Monash University, Melbourne, Australia
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Background: Persistence of HIV infection in resting CD4+ T-cells
remains the major barrier to HIV eradication. -Chun et al., Nat. Med., 1995; Chun et al., Nature, 1997; Finzi et al., Science, 1997; Brenchley et al., J Virol., 2004.
Infection of resting CD4+ T cells is difficult to establish in vitro due to multiple blocks in the viral life cycle.- Zack et al., J. Virol ., 1992; Zack et al., Cell, 1990; Bukrinsky et al., PNAS., 1992.
Latent infection can be established in resting CD4+ T-cells following incubation with multiple chemokines including the CCR7 ligand, CCL19 . - Saleh et al., Blood 2007; Cameron et al., PNAS 2010.
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chemokinesIn v
itro
Unactivated resting cellsResting CD4+ T-cell
Ex vivo tissue blocks
Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865; Saleh et al., Blood 2007; 110:416; Marini et al., J Immunol 2008; 181: 7713-20; Bosque and Planelle, Blood 2009; 113:58; Cameron et al., Proc Natl Acad Sci 2010 epub Sept 18
Infection of resting CD4+ T-cells
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CCL19 ligation activates cofilin and actin polymerisation
CXCR4 + gp120
CCR7 + CCL19
Yoder et al Cell 2008Cameron et al PNAS 2010
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Chemokine signalling pathways: PI3K
PI3K
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Chemokine signalling pathways: PLC & JAK/STAT
PLC
JAK/STAT
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What roles do these signaling pathways play in HIV integration in
resting T-cells?
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Hypothesis and aims
Hypothesis: HIV latent infection can be established in resting CD4+ T-cells through activation of specific chemokine signaling pathways.
Aim:To identify the signaling pathways critical for HIV integration in resting CD4+ T-cells.
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What is the role of the PI3K pathway?
WortmanninLY294002
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CCL19 (100nM)LY294,002 (50μM)
Wortmannin (100nM)PMA (200nM)
Total Akt
P-Akt
Merge
PI3K Inhibition of CCL19-induced Akt phosphorylation
- + - + - + - - - + + - - - - - - - + + - - - - - - - +
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PI3K pathway is critical for integration
Alu-LTR 2-LTR
100
1000
10000
100000
1000000
copi
es/m
illio
n ce
ll eq
uiva
lent
s
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Inhibition of PI3K has little effect on nuclear localisation (2LTR)
SC-514Bay 11-7082
SP600125
SB203580
PD980509
JNK
ERK
NF
B
NF
B
P38
P38ERK
JNKNFB
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Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR)
SC-514Bay 11-7082
SP600125
SB203580
PD980509
CCL
19+D
MSO
JNK
ERK
NF
B
NF
B
P38
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Infection with single round virus gave similar results
pNL4-3 env-Env deficient HIV-1
Env expression vector
pSVIII-HXB2 env
Co-transfected into 293T cells
LTR promoterSingle round Env
pseudotyped viruses
env-
(D. Purcell lab) (M. Churchill lab)
JNK
ERK
NF
B
NF
B
P38
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Blocking NFAT pathway has no effect on HIV nuclear entry
Cyclosporin Tacrolimus
PLC
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Blocking the NFAT pathway had no effect on integration
Cyclosporin Tacrolimus
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Summary
The Rho A pathway is important for HIV-1 nuclear entry in resting CD4+ T-cells.
Chemokines activate the PI3K pathway and this was critical for integration in resting CD4+ T-cells.
The JNK/ERK and NFB were the most important down stream proteins.
There was no effect of the PLC pathway on integration in resting CD4+ T-cells.
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Inhibition of Jnk and NF-kB eliminates integration
NFkB– Critical level required
for integration• Duverger J Virol 2009
– Transcription factors important for integration in active genes
• Felice, Plos One 2009
Jnk– Required for efficient
integrase cleavage via PIN 1
• Managanaro Nat Med 2010
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Conclusions
PI3K signaling is critical for HIV integration in chemokine treated resting CD4+ T-cells.
The most downstream critical proteins included both JNK and NF-B.
Strategies that target these pathways may potentially lead to novel interventions to block the establishment of latent infection.
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Future directions
To determine the role of the HIV LTR in facilitating integration using mutant viruses that lacks the common NF-kB binding sites in the LTR.
Identifying nuclear factors that are important for integration using a phospho-proteomic screen for kinase substrates activated by PI3K.
Selectively inhibit proteins that have been identified as important for HIV integration using siRNA.
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Acknowledgements
Department of Medicine, Monash University–Sharon Lewin–Paul Cameron–Georgina Sallmann
Burnet Institute–Anthony
Jaworowski–Melissa Churchill–Lachlan Gray