Transcript Slide 1
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:
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.
Infection of resting CD4+ T-cells Resting CD4+ T-cell Unactivated resting cells Ex vivo tissue blocks chemokines Eckstein et al,
Immunity
2001; 15: 671; Kreisberg et al.,
J Exp Med
110:416; Marini et al.,
J Immunol
2006; 203:865; Saleh et al., 2008; 181: 7713-20; Bosque and Planelle,
Blood Blood
2009; 113:58; 2007; Cameron et al.,
Proc Natl Acad Sci
2010 epub Sept 18
CCL19 ligation activates cofilin and actin polymerisation CXCR4 + gp120 CCR7 + CCL19 Yoder et al Cell 2008 Cameron et al PNAS 2010
Chemokine signalling pathways: PI3K
PI3K
Chemokine signalling pathways: PLC & JAK/STAT
JAK/STAT PLC
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?
Wortmannin LY294002
PI3K Inhibition of CCL19-induced Akt phosphorylation Total Akt
P-Akt Merge CCL19 (100nM) LY294,002 (50 μM) Wortmannin (100nM) PMA (200nM ) + + + + + + + +
PI3K pathway is critical for integration
Alu-LTR Un ac tiva te d IL2 /PH A CC L1 9 CC L1 9+L Y-29 CC 400 L1 2 9+Wo rt man an 2-LTR 1000000 100000 10000 1000 100 Un act ivated IL2/ PH A CC L19 CC L19 +L Y-29400 CC L19 2 +Wo rtm ana n
Inhibition of PI3K has little effect on nuclear localisation (2LTR)
ERK P38 JNK NF
B
SC-514 Bay 11-7082 SP600125 PD980509 SB203580
Inhibition of Erk1/2, Jnk and NF-kB eliminates integration (ALu-LTR) SC-514 Bay 11-7082 SP600125 PD980509 SB203580
Infection with single round virus gave similar results
pNL4-3 env-
Env deficient HIV-1
Env expression vector
pSVIII-HXB2
env env-
(D. Purcell lab) (M. Churchill lab) LTR promoter
Single round Env pseudotyped viruses Co-transfected into 293T cells
Blocking NFAT pathway has no effect on HIV nuclear entry
PLC
Tacrolimus Cyclosporin
Blocking the NFAT pathway had no effect on integration Tacrolimus Cyclosporin
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 NF B 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