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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 Presentation
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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
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.
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
CCL19 ligation activates cofilin and actin polymerisation
CXCR4 + gp120
CCR7 + CCL19
Yoder et al Cell 2008Cameron et al PNAS 2010
Chemokine signalling pathways: PI3K
PI3K
Chemokine signalling pathways: PLC & JAK/STAT
PLC
JAK/STAT
What roles do these signaling pathways play in HIV integration in
resting T-cells?
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.
What is the role of the PI3K pathway?
WortmanninLY294002
CCL19 (100nM)LY294,002 (50μM)
Wortmannin (100nM)PMA (200nM)
Total Akt
P-Akt
Merge
PI3K Inhibition of CCL19-induced Akt phosphorylation
- + - + - + - - - + + - - - - - - - + + - - - - - - - +
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
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
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
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
Blocking NFAT pathway has no effect on HIV nuclear entry
Cyclosporin Tacrolimus
PLC
Blocking the NFAT pathway had no effect on integration
Cyclosporin Tacrolimus
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.
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
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.
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.
Acknowledgements
Department of Medicine, Monash University–Sharon Lewin–Paul Cameron–Georgina Sallmann
Burnet Institute–Anthony
Jaworowski–Melissa Churchill–Lachlan Gray