Figure 2

Multiple cleavage products of A12L protein. A. BSC-40 cells were infected by VV WR and harvested at 24 hpi. Mock: cells alone, WR: VV WR-infected cell extracts. B. In order to determine whether A12L undergoes proteolysis, BSC-40 cells were infected with VV WR for 24 hours and incubated with rifampicin at concentrations of 0, 100, 150, 200, 300, and 400 μg/ml from left to right. As a positive control of drug induced-inhibition of VV proteolysis, p4b processing was demonstrated. C. Rifampicin-reversibility experiment. Cells were infected with VV and treated with rifampicin (150 μg/ml) at 5 hpi. The rifampicin was replaced with new infection media with and without the drug at 19 hpi to determine the effects of the drug on A12L protein processing for 12 hours. Mock (lane1): cells alone, Rif- (lane 2): rifampicin-free cell extracts harvested at 5 hpi, Rif- (lane 3): rifampicin-free cell extracts harvested at 19 hpi, Rif+ (lane 4): rifampicin-treated cell extracts harvested at 19hpi, Rif+/- (lane 5): rifampicin treated cells at 5 hpi and placed with new media without the drug at 19 hpi, Rif+/+ (lane 6): rifampicin treated cells at 5 hpi and replaced new media containing rifampicin at 19 hpi. Both Rif+/- and Rif+/+ were harvested at 31 hpi.