Figure 4

Protein analysis of viral particles generated by the 5, 6, and 7 plasmid systems. 293T cells were transfected with the 5 plasmid system (lanes 1 and 2), the 6 plasmid system (lanes 3 and 4), or the non-optimized 7 plasmid system (lanes 5 and 6), with (lanes 2, 4, 6) or without (lanes 1, 3, 5) Vif. Transfected cells were labeled with [³⁵S] methionine for 12 h, 48 h post transfection. Radiolabeled viral particles were pelleted, lysed, immunoprecipitated with anti-HIV serum and analyzed on 12.5% SDS-PAGE. The position of viral proteins are indicated, the boxed region shows the location of Vpr-RT/IN in which less accumulation of unprocessed Vpr-RT/IN can be seen for the 7 plasmid system as compared to the 6 plasmid system. Quantitative analysis of the CAp24 and Pr55Gag bands showed a 3 fold decrease in ratio of CAp24 to Pr55Gag for the 6 plasmid system and a 2 fold decrease for the 7 plasmid system (Cap24/Pr55Gag; 5 plasmid system 6.1 and 4.3, 6 plasmid system 1.6 and 1.8, 7 plasmid system 2.6 and 2.1, without and with Vif respectively). Lanes are not loaded equally. Mock, uninfected.