Figure 1.

Effect of proteasome inhibitors on HIV-1 release and Gag processing. HeLa cells transfected with HIV-1_NL4–3 were treated with 10 μM each of zLLL and LC or were left untreated during a pulse–chase experiment. Viral proteins were immunoprecipitated from the cell lysates, pelleted virions, and clarified supernatant, separated by SDS/PAGE, and analyzed by fluorography (A). Positions of the two major CA products, p24 and p25, are indicated by double arrows. The time course of virus release was calculated as the percentage of Gag (Pr55 and CA) present in the virus pellet relative to the total amount of Gag detected intra- and extracellularly (B). The rate of Pr55 processing was estimated by calculating the ratio of CA vs. Pr55 detected intracellularly at different time points (C). D shows a sucrose density gradient analysis of virus particles produced from HIV-1-infected A3.01 cells in the presence or absence of zLLL/LC. Individual fractions of the gradient were analyzed by Western blot by using HIV-1-specific antiserum. For studies on proteasome specificity, infected A3.01 (E) or transfected HeLa cells (F) were treated individually with proteasome inhibitors zLLL, LC, and epoxomicine or the control inhibitor zLL, respectively (final concentration 10 μM). After pulse–chase, virus release was calculated as above.