Antiviral activity of mutated siRNAs corresponding to common variants in gag and vif targets. siRNAs with single, double, or triple mutations were designed based on the common gag and vif sequence variants present in the Los Alamos HIV Sequence Database. Cells transfected with 200 nM of the indicated synthetic siRNA were challenged with HIV_IIIB virus on day 1 after transfection. Three days later, supernatants were collected for evaluation of p24 production by ELISA (the p24 level was 10 ng/mL for the control mock-treated cells and was inhibited to 0.5 ng/mL by wild-type [wt] gag or vif siRNA). Five days after infection, cells were stained for intracellular p24 and the antiviral activity of mutant siRNAs was assessed by comparing the percent of p24-positive cells in cultures treated with the mutated siRNA with that in wt siRNA-treated HeLa-CD4 cells (35% to 40% of mock-treated cells and 3% to 5% of wt siRNA-transfected cells stained for p24). Values represent means and SD of 2 independent experiments. The bars and circles represent the levels of inhibition obtained by intracellular staining for p24 and ELISA, respectively. The sequences of the mutant siRNAs are shown in Table 1.