FIG 2.

P122A and I124A mutations impair virus particle production and Gag processing. HeLa cells were transfected with WT and PPIP mutant proviral clones. At 24 h posttransfection, the cells were metabolically labeled with [³⁵S]Met/Cys. Viral particles were collected by ultracentrifugation. Cell lysates were immunoprecipitated with anti-HIV immunoglobulin (HIV-Ig). Viral proteins were separated by SDS-PAGE and quantified by phosphorimager analysis. (A) A representative gel. (B) Relative virus release efficiency was calculated as [virus-associated CA]/[total (cell plus virus) Gag + CA], with the WT value set at 100; error bars indicate standard deviations (SD); n = 3 independent experiments. (C) Gag processing efficiency in cell lysates was calculated as CA/(CA + Pr55Gag). Error bars indicate SD; n = 3 independent experiments. (D) The level of unprocessed Pr55Gag in virions collected from 293T cells was assessed by western blotting and calculated as Pr55Gag/(Pr55Gag + CA). Sample loading was adjusted to reflect the decreased particle production of the P122A and I124A mutants (a representative gel is shown on the left; quantitation indicated on the right). Error bars = SD; n = 3 independent experiments. (E) Percentages of CA-SP1 calculated as CA-SP1/(CA-SP1 + CA). 293T cells transfected with WT and mutant clones were incubated in the presence of dimethyl sulfoxide (DMSO) or 100 nM maturation inhibitors (BVM or the 7m or 7r analogs) and were metabolically labeled in [³⁵S]Met/Cys. Radiolabeled virions were collected and viral proteins separated by SDS-PAGE. WT protein bands were exposed to a phosphorimager screen for 1 day; mutant viral samples were exposed for 4 to 5 days. Error bars indicate SD; n = 3 independent experiments. (F) HeLa cells were transfected with PR⁻ molecular clones. Virus release efficiency was calculated as virion Pr55Gag/total (cell plus virus) Pr55Gag. Error bars indicate SD; n = 3 independent experiments.