PAP does not alter HIV-1 virus particle composition.

<p>Virus particles were collected from media of 293 T cells transfected with the pNL4-3 proviral clone (5 µg) and 0.5 µg 3x-Flag-PAP, 3x-Flag-PAPx or pcDNA3 vectors. (A) Particles were purified by ultracentrifugation through a 20% sucrose cushion and equivalent amounts of purified virus (100 ng) were resolved on 10% SDS-PAGE. Viral proteins were then transferred to nitrocellulose and probed with antibodies specific for RT (1∶2,500), gp120 (1∶5,000) and p24 CA (1∶5,000) proteins. (B) RNA was isolated from equivalent amounts of purified virus particles (100 ng) and analyzed by RNase protection assay using an antisense riboprobe complementary to the gag ORF (nt 600–820). A fraction of the purified viruses was used for immunoblot analysis of p24 CA to control for equal virus pelleting from each sample (lower panel). Blots are representative of five independent experiments performed in triplicate.