FIG. 5.
Further evidence supporting a role for cathepsin B in the membrane fusion step of infection. (A) Cathepsin B−/− MEFs are not sensitive to inhibition by CA-074 Me in the range that is selective for inhibition of cellular cathepsin B. Quadruplicate samples of B−/− and control B+/+ MEFs were preincubated with 0, 1, 2.5, 5, 7.5, and 10 μM CA-074 Me for 1 h prior to the addition of replication-defective virus plus the inhibitor; 6 h later, the cells were fixed, stained, and then scored by light microscopy. Similar results were obtained in two additional experiments. One hundred percent infection (no CA-074 Me) was 1 × 103 ± 0.1 × 103 for B+/+ cells and 4.8 × 102 ± 0.1 × 102 LacZ TU/ml for the poorly susceptible B−/− cells. The error bars indicate standard deviations. (B) Addition of purified exogenous cathepsin B increased infection of B−/− MEFs. Triplicate samples of B−/− and B+/+ cells were exposed to virus mixed with the indicated amounts of purified cathepsin B. Similar results were observed in two additional independent experiments. One hundred percent infection (no cathepsin B added) was 3.8% ± 0.8% of B+/+ cells infected and 0.8% ± 0.2% of B−/− cells. The standard deviations are not visible for the 100-ng B+/+ and 250-ng B−/− samples because their error bars were smaller than the symbol for the data point. (C) Addition of exogenous cathepsin B overcame the inhibition of fusion resulting from CA-074 Me. Quadruplicate wells of NIH 3T3 cells were preincubated with 50 μM CA-074 Me for 2 h to irreversibly inhibit endogenous cathepsin, and then the cells were rinsed once in DMEM, and virus plus the indicated amounts of purified cathepsin B were applied to the cells for 6 h. A similar restoration of syncytium induction was seen in an independent experiment.