Fig. 8

Vpr-induced DSB stimulates viral infection in resting macrophages. a Vpr induced DSBs in resting macrophages. Differentiated MM-6 cells were infected with NL4-3/D64A virus for 1 day, and then subjected to neutral comet assay. Representative images of each comet are shown (upper panel). The relative Olive-tail moment was evaluated (lower panel). Error bars indicate ± SEM calculated based on data obtained from more than three independent experiments. Data from non-infected cells (−), Vpr-proficient virus (R+), and Vpr-deficient virus (R−) are shown. **P < 0.05. b Involvement of Topo1 in Vpr-induced DSB. Neutral comet assay was performed as in a after 3 days of Topo1 targeting siRNA transfection. c DSB induced by rVpr was blocked by down-regulation of Topo1. Three days after transfection of indicated siRNA, cells were treated with rVpr (100 ng/ml, 16 h). d Integration of proviral DNA was blocked by down-regulation of Topo1. MM-6 cells were infected with Vpr-proficient (R+) or deficient (R−) NL4-3 virus after transfection with siRNA, and the integration rate was quantitated by Alu-gag two-step nested qPCR at 2dpi; relative integration rates are shown. e Vpr-dependent increase of viral infectivity required Topo1 in MDMs. MDMs from two healthy donors were infected with Vpr-proficient (R+) or deficient (R−) NL4-3 virus 2 days after transfection with Topo1 targeting siRNA, and the integration rates were quantitated as in d at 2dpi