FIG. 1.

Effect of cathepsin inhibitors and siRNA on infection of Vero cells by VSV-GP. (A) Cells were pretreated for 2 h at 37°C with vehicle alone (V), CatB inhibitor (B), or CatL inhibitor (L) at low concentrations (0.5 μM CatB inhibitor, 1 μM CatL inhibitor) and high concentrations (1 μM CatB inhibitor, 4 μM CatL inhibitor). Cells were washed and infected with VSV-GP and VSV-G pseudovirions at approximate multiplicities of infection (MOI) of 0.2 to 0.4 and 0.5 to 1.0, respectively, in the presence of fresh inhibitors. Twenty-four hours postinfection, the cells were fixed and the percentage of GFP-positive cells was determined by flow cytometry. Results shown are the averages of normalized data from three to six experiments, where the average percent infection for vehicle-treated cells was 31 ± 19 for VSV-GP and 56 ± 10 for VSV-G. Error bars indicate standard errors, and asterisks indicate a statistically significant deviation from the mean relative to vehicle-treated cells at a confidence level of greater than 99%. (B and C) Cells were transfected with a nontargeting control siRNA oligonucleotide (Con) or siRNA oligonucleotides targeting CatB and/or CatL. Seventy-two hours posttransfection, the cells were either lysed for Western blotting and activity assays (B) or infected as described for panel A (C). (B) Representative blot of CatB and CatL expression in siRNA-treated cells and the average results from five activity assays ± the standard deviation. Results shown in panel C are the averages of normalized data from three to six experiments, where the average percent infection for control siRNA-treated cells was 23 ± 14 for VSV-GP and 53 ± 6 for VSV-G. Error bars indicate the standard errors, and asterisks indicate a statistically significant deviation from the mean relative to control siRNA-treated cells at a confidence level greater than 99%.