Figure 7

Vpr mimics DNA damaging agents, and enhances the IN-CA independent macrophage infection. (A) Vpr induces DNA damage cellular signals in MDMs. HT1080 cells or MDMs were infected with VSVG-pseudotyped R– virus (NL-Luc-E(−)R(−)) or R+ virus (NL-Luc-E(−)), and then analyzed immunochemically. Bars = 10 μm. (B) Effect of RAL on the infectivity of WT and D64A viruses. MDMs were infected with WT or D64A viruses in the presence of AZT or RAL. The cells were harvested at 48 hpi and subjected to luciferase assay. Relative luciferase activity values to WT R– infectivity are shown. Black circles, WT; gray circles, D64A; ND, not detected. Error bars, s.d. of triplicate assays. (C) Effects of Vpr on the integration of viral DNA into the host genome. Serum-starved HT1080 cells were infected with VSVG-pseudotyped IN WT or D64A mutant virus with or without Vpr. After 48 h, infected cells were subjected to qPCR analysis. Error bars, s.d. of triplicate assays. *P < 0.05. (D) HIV-1 replicates in MDMs in the presence of RAL. Replication-competent NL4-3 with an intact env gene derived from R5-tropic ADA viruses (NL-ADA, NL-ADA-R(−), NL-ADA-IN-D64A, and NL-ADA-IN-D64A-R(−)) were infected. Then, copy numbers of HIV-1 RNA in the conditioned medium was quantified by RT-qPCR. E and F) Positive effects of Vpr on infection of D64A virus into MDMs. Primary cells and cell lines were infected with IN WT or D64A mutant virus with or without Vpr. Cells were harvested at 48 hpi and subjected to luciferase assay. (E) Relative luciferase activity values to WT R– infectivity are shown. White bars, WT/R–; light gray bars, WT/R+; dark gray bars, D64A/R–; black bars, D64A/R+. Error bars, s.d. of triplicate assays. (F) Fold increase of R+ virus infectivity to R– virus. White bars, WT; black bars, D64A.