FIG. 2.

Requirements for the release of particles containing SV5 proteins from transfected cells. (A) 293T cells were either infected with rSV5 or transfected with the indicated plasmids. At 24 h p.i. or p.t., cells were radiolabeled for 16 h with [³⁵S]Promix, followed by collection of both cells and culture media. Culture media was clarified by low-speed centrifugation, centrifuged through 35% sucrose cushions, and separated on sucrose flotation gradients into two equal fractions. The bottom fraction was discarded and proteins in the top (floated) fraction were analyzed by immunoprecipitation. Cells were disrupted by Dounce homogenization and centrifuged at low speed to remove nuclei and debris. SV5 proteins were immunoprecipitated from samples as described in Materials and Methods, and polypeptides were analyzed by SDS-PAGE on 10% gels. (B) Budding efficiency was quantified as the fraction of SV5 M protein detected in the media compared with the total SV5 M protein in the media plus the cell lysate. Values represent averages from three experiments, and the standard deviations are indicated.