Figure 5

Loss of Spt6 or PAAF1 induces aberrant HIV-1 transcript synthesis. (A) Extracts of HeLa-LTR-luc cells transfected with siRNA against PAAF1, Spt6 or a control (Scr) and subsequently transduced with GST-Tat (Tat) as indicated were analyzed by IB using the indicated antibodies, Luciferase activity that was normalized to protein content, and RT-Q-PCR using Luc primers, which was normalized to the quantity of GAPDH mRNA in each sample. Values obtained for siScr samples were set to 1. (B) Reverse transcripts in A were analyzed by Q-PCR with primer pairs spanning the transcript. A schematic diagram of primer locations is shown above the graph. Transcript is shown as a black line containing the TAR RNA stem-loop at the 5' end. Values were normalized to the amount of GAPDH mRNA for each sample, and the value obtained for the TAR region was set to 1 for each condition. Graphs show mean +/- SD (n = 3). (C) Increase of HIV-1 transcripts following ablation of PAAF1 can be partially suppressed by exogenous Spt6 expression. HeLa-LTR-luc cells were transfected with Scr or PAAF1 siRNA and HA-Spt6 or mock expression vector, as indicated. Total RNA was isolated and analyzed by RT-Q-PCR using primers in the coding region. Graph shows mean +/- SD (n = 3).