Vpu contributes to p53-mediated apoptosis during infection. MOLT-3 T cells or K-562 cells were infected with an MOI of 1 with indicated VSV-G–pseudotyped HIV-1 virus. (A) Total cell death was measured after 48 hours of infection of MOLT-3 cells by PI staining by FACS: PI (FL-2H), (left) live cells (PI⁻), (right; region R1): dead cells (PI⁺). p53-mediated apoptosis was inhibited by treating cells with 30μM Pifithrin-α for 24 hours (samples 5-8) as described in “Infection by HIV-1 pNL4-3 or HIV-1 mutants.” Percentage of cell death is indicated for each sample. (B) Total cell death (region R1) was measured 48 hours after infection of MOLT-3 and K-562 cells with indicated viruses. After 48 hours of infection cell lysates were prepared, and immunoblotting was done with indicated Abs (C-D). Cells were evaluated for the extent of HIV-1 infection by intracellular p24 staining with primary p24 rabbit Ab (NIH) followed by secondary anti–rabbit Ab (phycoerythrin conjugated). (E) Representative pictures showing comparable infection efficiency of the various subtype B viruses in MOLT-3 cells by intracellular p24 staining.