FIG 2.

BBC does not affect structural protein synthesis or processing. (A) Steady-state structural protein production. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with dimethyl sulfoxide (DMSO) or BBC from 4 to 8.5 hpi. Cells were then solubilized in Triton X-100-containing lysis buffer and centrifuged to obtain detergent-insoluble pellets and lysates. Samples were analyzed by SDS-PAGE and Western blotting (WB) using polyclonal antibody (PAb) to E2 and E1 and monoclonal antibody (MAb) to Cp. Histone H3 and β-tubulin served as loading controls for the detergent-insoluble and cytoplasmic extracts, respectively. Treatment labels are as follows: D, 0.1% DMSO; 10, 10 μM BBC; 20, 20 μM BBC; 50, 50 μM BBC. (B) Pulse-chase analysis of protein synthesis and processing. BHK-21 cells were infected with ppSFV at an MOI of 10, treated with DMSO or BBC at 4 hpi, pulse labeled for 30 min with [³⁵S]methionine/cysteine at 5 hpi, and chased for the indicated times, all in the continued presence of DMSO or BBC. At each time point, the cells were lysed, and the envelope proteins were immunoprecipitated with a PAb to E2 and E1. Samples were analyzed by SDS-PAGE and fluorography. (C) WB analysis of E2 to Cp ratio of pelleted particles. BHK-21 cells were infected with ppSFV at an MOI of 10 and treated with 0.1% DMSO or 20 μM BBC at 4 hpi, and the cell media were collected at 8.5 hpi. Particles were pelleted through a 20% sucrose cushion and resuspended in buffer. Samples were analyzed by SDS-PAGE and WB using MAb to E2 and PAb to Cp. The results shown in panels A, B, and C are representative of three independent experiments.