287 PI3-Kinase Regulates HIV-1 Replication Following Viral Entry in Primary CD4+ T-lymphocytes Fleur Francois, Mary E. Klotman* Mt Sinai Sch of Med, New York, NY
Background: Previously, we have shown that HIV-1 gp120 activates the PI3-kinase pathway and that basal and induced PI3-K activity is required for HIV-1 replication within primary CD4+ T-cells and macrophages. Here, we determine the stage of HIV replication at which PI3-kinase activity is required for infection.
Methods: To delineate which steps of HIV replication are regulated by PI3-K, cells were either treated with PI3-K inhibitor LY294002 before or after a 2 hr incubation with X4 and R5-pseudotyped pNL4-3-Luc-R-E- virus and removal of the unbound virus. R5 and X4 entry inhibitors, TAK779 and AMD3100, and reverse transcriptase inhibitor (AZT) were included as controls. Semi-quantitative PCR amplification of R/U5 and LTR/gag was used to determine whether LY294002 inhibited infection before or after reverse transcription. To test whether LY294002 inhibited HIV transcription the inhibitor was added following a 24-hr incubation with VSV-pseudotyped pNL4-3-Luc-R-E- virus.
Results: LY294002 and AZT decreased infection of CD4+ T-cells with both R5 and X4-pseudotyped HIV-1 luciferase viruses regardless of whether they were added before or after virus entry. In contrast, HIV-1 virus entry inhibitors TAK779 and AMD3100 lost their ability to protect CD4+ T-cells from infection when added after virus entry. LY294002 did not decrease the amount of HIV-1 RT products R/U5 and LTR/gag obtained following infection with HXB2 or JRFL-pseudotyped viruses. However, when the inhibitor was added after viral integration had occurred, no inhibition of HIV replication was observed.
Conclusions: Our studies show that PI3-Kinase inhibition suppresses HIV infection post-viral entry and post-reverse transcription but prior to HIV-1 gene expression. This type of host-virus interaction has implications for anti-HIV therapeutics that target cellular signaling machinery.
