Fig. 6

Impact of UNG2 and RPA32 on HIV-1 replication in macrophages. a–c Wild-type (a) or Δvpr (b) replication-competent viruses were produced in shLuc- (black curves and bars), shUNG2- (red curves and bars) or shRPA32- (green curves and bars) transduced 293T cells, normalized for p24, and then used for infection in duplicate of MDMs from 3 different healthy donors. In a and b, aliquots of MDM cell culture supernatants were collected 4 and 8 days after infection for p24 quantification. The individual kinetics of replication in PBMCs from the three healthy donors are shown. In c, results are expressed as the percentage of p24 production at each time point relative to that of MDMs infected with wt or Δvpr viruses produced in shLuc-transduced (black bars) cells. Values are the means of two independent experiments performed on MDMs from the two donors. d Virus infectivity in MDMs. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect MDMs from three different donors. The percentages of GFP-positive infected cells were then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in shLuc-transduced (black bars) 293T cells. e Replication-competent viruses were produced in shLuc-, shUNG2- or shRRA32-transduced 293T cells, normalized for p24, and then used for infection of MDMs from three different donors. MDM samples were collected 72 h after infection, subjected to DNA purification, and total viral DNA was quantified via qPCR using specific primers for U5-gag. Results are expressed as the percentage of total viral DNA relative to that of MDMs infected with viruses produced in shLuc-transduced (black bar) cells. f Double-depletion of UNG2 and RPA32 expression in virus-producing 293T cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2 or Luciferase and containing the gene for puromycin resistance, and with lentiviral vectors expressing shRNA against RPA32 or Luciferase and the GFP reporter gene. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32 and anti-β-actin antibodies. g Replication-competent viruses were produced in shLuc/shLuc-GFP (black bar), in shUNG2/shLuc-GFP (red bar) or in shUNG2/shRPA32-GFP (red and green hatched bar) 293T cells, normalized for p24, and then used for infection of MDMs from three different healthy donors. The concentration of p24 after 8 days of infection was expressed as the percentage of p24 production relative to that of MDMs infected with viruses produced in shLuc-transduced (black bar) cells. Error bars represent the SEM. Statistical significance was determined using Students t test (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001)