Figure 7

Depletion of eIF5A by siRNA inhibits gene expression from HIV-1 molecular clone. A. Depletion of eIF5A. 293T cells were transfected with 50 nM of eIF5A-1 siRNA (5A) or control siRNA (C, with no known complementary sequence in the human genome). Total RNA was isolated from transfected cells at the times indicated and analyzed by RPA using probes for eIF5A-1 or GAPDH mRNA (panel A). B. Effect of siRNA on HIV gene expression. siRNA-transfected cells were co-transfected with pNL4-3-LucE^- and pCMV-Ren at 1, 3, 4, 5, 6 and 7 days after siRNA transfection and harvested 24 hr later for luciferase assays (top panel) as in Fig. 3. Relative FF/Ren luciferase expression at each time point is shown as a percentage inhibition of the control ratio (siC) obtained in the presence of si5A (triplicate measurements ± SD). Parallel cultures were analyzed for eIF5A and actin by immunoblotting (bottom panels). C. Lack of synergy between siRNA and drugs. siRNA-transfected 293T cells were additionally transfected with pNL4-3-LucE^- and pCMV-Ren 4 days later and simultaneously treated with CPX or DEF as indicated. Luciferase assays were analyzed as in Fig. 3. Immunoblots for eIF5A and actin are shown in the lower panels for days 3 and 4 after siRNA transfection.