FIG. 4.

Effect of Tetherin depletion on HIV-1 particle release from nonpermissive HeLa cells. (A) HeLa cells were transfected with nontargeting siRNA (siGENOME control siRNA [catalog no. D-001210-02-20; Dharmacon]; lanes 1 to 3) or specific siRNA against Tetherin (ON-TARGET plus SMART pool [catalog no. L-011817-00; Dharmacon]; lanes 4 to 6). Subsequently, cells were mock-transfected (M; lanes 1 and 4) or transfected with the proviral plasmid HxBH10vpu− (−; lanes 2 and 5) or HxBH10vpu_wt (wt; lanes 3 and 6) as indicated. Cells and supernatants containing viral particles were harvested 24 h posttransfection, and lysates were analyzed by Western blotting. Cell lysates were analyzed to detect Gag products and endogenous cellular Tetherin by using anti-p24 antibodies (the asterisk denotes a nonspecific band used as an internal loading control). Virus lysates were analyzed for the presence of p24 by using specific antibodies. (B) Densitometric quantification of HIV-1 release efficiency after endogenous Tetherin depletion. HIV-1 particle release efficiency was evaluated as described in the legend to Fig. 1B. The release efficiency of HxBH10vpu_wt was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of results from two independent experiments.