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Fig. 3 | Retrovirology

Fig. 3

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. 3

Impact of UNG2 and RPA32 on HIV-1 replication in Jurkat T cells. a Depletion of UNG2 and RPA32 in Jurkat cells. Cells were transduced with lentiviral vectors expressing shRNA against UNG2, RPA32 or Luciferase. Lysates from shRNA-transduced cells were analyzed by Western blot using anti-UNG2, anti-RPA32, anti-RPA70 and anti-β-actin antibodies. be Virus replication in UNG2- or RPA32-depleted Jurkat cells. Replication-competent viruses were produced in UNG2- (B and D) or RPA32- (c, e) depleted cells or in control shLuc-transduced 293T cells, normalized for p24, and then used for infection of shLuc-, shUNG2- or shRPA32-transduced Jurkat cells. Aliquots of cell culture supernatant were collected 2, 4, and 8 days after infection for p24 quantification. In b, c, the kinetic of replication shown is representative of four independent experiments. In d, e, values are the means of the four independent experiments. Results are expressed as the percentage of p24 production at each time point relative to that of shLuc-transduced Jurkat cells infected with viruses produced in shLuc-transduced 293T cells. f Virus infectivity in UNG2- and RPA32-depleted cells. 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 Jurkat 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 shLuc-transduced 293T cells and measured on shLuc-transduced Jurkat as target cells. Error bars represent the SEM. Statistical significance was determined by using the Students t test (ns, p > 0.05; *p < 0.05; **p < 0.01)

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