Fig. 3.

Effect of DDX17 knockdown on HIV-1 splicing and Gag. (a) RT-qPCR analysis of unspliced, (b) partially and (c) fully spliced HIV-1 mRNA respectively. Cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI. Data shown are representative of three independent repeats, with triplicate samples for each siRNA. Data have been normalized to siControl. Error bars represent ± SEM. Values are scored as a fold-change relative to that of siControl-treated cells. Statistical significance *P < 0.05. (d) Confocal microscopy results showing intracellular HIV-1 Gag distribution following siRNA treatment and rescue in HeLa M cells. From left to right, DAPI, Gag and merged columns. Cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI, with or without increasing concentrations of siDDX17 rescue expressor construct. Cells were harvested 48 h post-co-transfection and dual stained for DAPI and Gag. (e) Western blot showing the effect of DDX17 knockdown on cellular CA-p24. Cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI, with or without increasing concentrations of siDDX17 rescue expressor construct. (f) Western blot showing the effect of DDX17 knockdown on Rev, Nef and Vif levels. Cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI, with or without increasing concentrations of siDDX17 rescue expressor construct. (g) Western blot showing the effect of DDX17 knockdown and rescue on Env and Vpu levels. (h) Western blot showing the effect of DDX17 knockdown on Tetherin levels.