Vpr is required for the 2nd round of infection. (A) Schematic illustration of the 1st round viral replication assay. WT or ΔVpr HIV-1 luciferase (Luc)-reporter viruses were produced by transfection of 293T cells with pNL-ΔGag plus pNL-Luc-ΔEnv, or pNL-ΔGagΔVpr plus pNL-Luc-ΔEnvΔVpr vectors. After normalization by p24Gag ELISA, equal amounts of viruses were used to infect indicated cells. Two days later, viral infectivity was determined by measuring the intracellular luciferase activity (B), or viral production from the supernatant by p24Gag ELISA (C). (D) Schematic illustration of the 2nd round viral replication assay. Indicated cells were infected with VSV-G pseudotyped WT or ΔVpr HIV-1 (NL4-3) by spinoculation. Three days later, virions were collected from supernatants, and equal amounts of virions were used to infect the HIV indicator TZM-b1 cells. Thirty-six hours later, viral infectivity was determined by measuring the intracellular luciferase activity (E). The standard errors (S.E.) in (B) and (E) were calculated from three independent experiments.