MOAA0102 - Oral Abstract Session
The PI3K signaling pathway is critical for HIV integration in latently infected resting CD4+T-cells
Presented by Suha Saleh (Australia).
S. Saleh1, P. Cameron1,2, G. Sallmann1, A. Joworowski1,3, S. Lewin1,2,3
1Monash University, Medicine, Melbourne, Australia, 2Infectious Disease Unit of Alfred Hospital, Melbourne, Australia, 3Burnet Institute, Melbourne, Australia
recently showed that latency can be established in resting CD4+ T cells
following incubation with chemokines that bind to CCR7, CXCR3 and CCR6 receptors
that are highly expressed on resting CD4+ T cells. Receptor ligation allowed
for increased efficiency of nuclear localization and integration of HIV DNA in
resting CD4+ T-cells. We hypothesised that activation of specific signaling
pathways including the phosphoinositol 3 kinase (PI3K) allowed for efficient
nuclear localization and viral integration in resting CD4+ T-cells.
Methods: Resting CD4+ T-cells were incubated
with either CCL19, PHA/IL-2 or unactivated for 24 hours and then infected with
HIV-1 in the presence or absence of pharmacological inhibitors of the PI3K
pathway. q PCR was used to quantify 2-LTR circles (as a marker of nuclear
localization) and integrated HIV DNA at day 4 post-infection. To detect activation
of the PI3K/AKT pathway, cells were stimulated with CCL19 +/- the inhibitors
and analysed Akt phosphorylation by immunoblotting.
Results: Infection of cells in the presence of
CCL19, PHA/IL-2 or no activation led to a mean of 11 916, 71 000 and < 300 integrated
copies/million cells and 25 333, 154 500 and 74 132-LTR copies/million cells
respectively. Incubation of cells with CCL19 and the pan PI3K inhibitors
Ly294002 and wortmannin, or specific down streams inhibitors PD98059 (ERK1/2),
SP600125 (JNK), SC-514 & BAY11-7082 (NF-kB),
completely eliminated integrated DNA but only slightly changed 2-LTR.
Inhibition of the AKT pathway with SB203580, a compound inhibiting p38 kinase
activity had no effect on viral integration. Ly294002 and wortmannin were active as confirmed by complete blocking of Akt phosphorylation in
response to CCL19.
Conclusion: In our model of
chemokine induced latency, efficient integration in resting CD4+ T-cells was
mediated by the PI3K pathway. Strategies that target these pathways may potentially
lead to novel interventions to block the establishment of latent infection.
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