Figure 5. Vpr is sufficient for the cell cycle arrest and suppression of cellular translation.

(A) HEK 293 cells were transfected with IRESgfp (Empty plasmid), bicistronic VprIRESgfp expression plasmid that encodes Vpr or indicated substitution mutants, or IRESgfp plus Vif expression plasmid. GFP-positive cells were sorted and metabolically labeled with [³⁵S]-cysteine/methionine for one hour, followed by TCA precipitation and scintillation. The histogram presents average TCA precipitable counts of the incorporated [³⁵S] and the standard deviation from three independent experiments. Asterisk indicates a significant reduction in de novo protein synthesis (p = 0.0019) in response to Vpr. Propidium iodide staining intensity and ModFit analysis determined the number and percentage of cells in the G1 or G2/M phase of the cell cycle. Percentages are summarized below the plots. (B) Equivalent whole-cell extracts were immunoblotted with HA antibody to detect epitope tagged Vpr, and antiserum against Vif, PARP and β-Actin, respectively.