Fig. 3.

BRET2 analyses. (A) 293T cells were mock transfected or co-transfected with GagPol PR+ or GagPol PR− and hGFP2-p2/p7-hRLuc. Increasing concentrations (0.5, 1, and 1.5 μg) of a Vif expressor were included with GagPol PR+ and hGFP2-p2/p7-hRLuc (last three lanes). Cells were harvested and blotted for hGFP2 and Gag (not shown). Overexpression of Vif caused a dose-dependent accumulation of hGFP2-p2/p7-hRLuc fusion protein precursor indicating an inhibition by Vif on PR activity at the p2/p7 site. Vif expression inhibited PR activity as determined by BRET2 (see B) to almost 85% that obtained in the PR− context. (B) 293T cells were transfected as above except several controls were included (hRLuc and hGFP2 expression alone). The transfection conditions are indicated above and below the histogram. The threshold BRET2 value is shown by the dashed line and the error bars represent the mean ± S.E.M. from at least five independent experiments, except for the last two conditions when the Staufen double stranded RNA-binding mutant protein (dsRBDmut) and TRBP proteins are expressed (1.5 μg) for which the error bar represents the standard deviation calculated from two independent experiments.