Vpr mutagenesis analyses. (A) Analysis of Vpr protein expression in viral producer cells. HIV-1 proviral vectors expressing indicated Vpr proteins were transfected into 293T cells, and Vpr expression was determined by Western blotting. The amounts of actin were used as loading control. (B) Analysis of cell cycle. SS and NKR cells were infected with HIV-1 expressing indicated Vpr proteins. After two days, intracellular Gag proteins were stained with fluorescent antibodies and intracellular DNAs were stained with propidium iodide. The Gag-positive population was sorted and analyzed for cell cycle profiles by flow cytometry. The G2/G1 ratios were calculated by dividing the proportion of cells in G2 and M phases with that in G0 and G1 phases. (C) Replication of HIV-1 bearing Vpr mutations. SS, H9, N8-SP, and As2O3-treated N2-NP [N2 (+As)] and NKR [NKR (+As)] cells were infected with HIV-1 expressing indicated Vpr proteins. Viral replication was detected by p24Gag ELISA. All experiments were repeated at least twice and consistent results were obtained.