FIG. 1.

Relationship of cell confluence to the effect of Vpu on particle release. 293T cells were plated in 35-mm wells at 100, 75, 50, 25, and 10% confluencies. Each well was labeled with 50 μCi of [³H]thymidine/ml overnight. The next day, equal numbers of cells were lysed on glass filters (Fisher) and washed three times with radioimmunoprecipitation buffer, and [³H]thymidine incorporation was determined. The data at the bottom show levels of ³H disintegrations per minute at each confluence, as well as the fold above 100% confluence. A duplicate set of cells grown at the same confluencies were used to assess Gag protein synthesis and viral particle release as described previously (3). For infection-transfection, cells were first infected with a recombinant vaccinia virus expressing T7 polymerase, vTF7-3, at a multiplicity of infection of 10. At 30 min postinfection, cells were transfected with 10 μg of total DNA by use of Lipofectamine. At 5 h posttransfection, the medium was removed and 50 μCi of Tran³⁵S label/ml was added. Cell lysates and supernatants were harvested and prepared for immunoprecipitation analysis with anti-p24 (CA) antisera. The proteins were examined by SDS–12.5% PAGE. pTM3 is a plasmid lacking gag and pol sequences. The top panel shows levels in cell lysates. The bottom panel shows the amount of Gag released into supernatants at each confluence.