FIG. 1.

Vpr can be efficiently packaged in trans in virions and induces cell cycle arrest in the G₂ plus M phase. (A) Ten nanograms of p24^gag from concentrated virus preparations was analyzed for the presence of trans Flag-tagged Vpr in Vpr(+)^trans viruses. The blots were probed with a mouse monoclonal antibody raised against the Flag tag. Vpr(+)^cis viruses that expressed Flag-tagged Vpr in cis were used as positive controls. Vpr(−) viruses that did not contain virion-associated Vpr were used as negative controls. p24^gag was used as a loading control. (B) HeLa cells (5 × 10⁴) were seeded 24 h prior to infection and then mock infected or infected with an equivalent number of EGFP transduction units of Vpr(−) and Vpr(+)^trans viral stocks. Two days postinfection, cells were harvested and stained with propidium iodide. The percentage of cells at the G₁ or G₂ plus M phase was determined after analysis of the cell cycle profiles with ModFit software, and the G₂-plus-M/G₁ ratio was then calculated. The left and right black peaks represent the relative numbers of cells in the G₁ and G₂ plus M phases, respectively. The results shown are representative of three independent experiments in which similar results were obtained.