FIG. 2.

BHV-1 gM can be immunoprecipitated from virions, infected cells, virion envelopes, and the membranes but not the cytosol of infected cells. (A) MDBK cells were infected (Inf) at an MOI of 10 or mock infected and labeled with [³⁵S]methionine and -cysteine for 16 h beginning 6 h after infection. Labeled virions were semipurified by centrifugation through a 30% sucrose cushion. The cells and virions were lysed with NP-40 and sodium deoxycholate. Samples were immunoprecipitated with the gMC or GST antibody (Ab), treated at 56°C with SDS-PAGE sample buffer in the presence of the reducing agent DTT, analyzed by SDS-PAGE on a 12% gel, and autoradiographed. (B) Metabolically radiolabeled virions were lysed in NP-40 and sodium deoxycholate and either loaded directly on the gel (−) or immunoprecipitated with gMC antibody (+). Another sample of virus was treated with detergents, and the envelope fraction was cleared of nucleocapsids by centrifugation over a 30% sucrose cushion and either loaded directly on the gel (−) or precipitated with gMC antibody (+). Samples were treated with DTT at 56°C, analyzed by SDS-PAGE on a 12% gel, and autoradiographed. (C) Cells were infected and labeled as for panel A. Cell membranes were obtained from cells disrupted in a Dounce homogenizer, centrifuged at low speed to remove cell debris, pelleted at 12,000 rpm, washed with homogenizing buffer, and repelleted at 40,000 rpm. Lysates of total cells, the 40K supernatant from washed membranes (40KSupe), and the washed membranes themselves were immunoprecipitated with antibody against GST or gM. Precipitates were treated with DTT at 56°C, analyzed by SDS-PAGE on a 12% gel, and autoradiographed.