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Fig. 2 | Retrovirology

Fig. 2

From: Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination

Fig. 2

Impact of UNG2 and RPA32 depletion on HIV-1 replication in HeLa-CD4 cells. a Depletion of UNG2 and RPA32 in 293T (left panels) and HeLa-CD4 (right panels) cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase (Luc) used as a control. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. b, c Virus replication in UNG2- or RPA32-depleted cells. Replication-competent viruses were produced in UNG2-, RPA32-depleted or in control shLuc 293T cells, normalized for viral p24, and then used for infection of UNG2-depleted (red line and bars), RPA32-depleted (green line and bars) or control shLuc (black line and bars) HeLa-CD4 cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b, the kinetic of replication shown is representative of four independent experiments. In c, results are the means of the four independent experiments and are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses. d Virus infectivity. Wild-type GFP reporter viruses were produced in shUNG2-, shRPA32- or shLuc-transduced 293T cells, normalized for p24, and then used to infect shUNG2-, shRPA32- or shLuc-transduced HeLa-CD4 cells as indicated. The percentage of GFP-positive infected cells was then measured by flow cytometry 60 h later. Viral infectivity was normalized to that of viruses produced in control 293T cells and measured on control HeLa-CD4 as target cells. e Quantification of total viral DNA. Infected HeLa-CD4 cells were collected 7 h after infection, subjected to DNA purification, and the total viral DNA was quantified by qPCR using specific primers for U5-gag. Results are expressed as the percentage of total viral DNA relative to that of shLuc-transduced HeLa-CD4 cells infected with control viruses produced in shLuc-transduced 293T cells. Values are the means of at least three independent experiments. Error bars represent 1 SEM (standard error of the mean). Statistical significance was determined using Students t test (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001)

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