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. 2012 Mar;86(5):2533–2544. doi: 10.1128/JVI.05163-11

Fig 4.

Fig 4

Impact of UNG2 depletion on virus infectivity. (A) and (B) Depletion of UNG2 in virus-producing (293T) and target (HeLa-CD4) cells. 293T or HeLa-CD4 cells were transduced with lentiviruses expressing shRNA against either UNG2 or luciferase used as a control. In panel A, lysates from shRNA-transduced 293T and HeLa-CD4 cells were analyzed by Western blotting using anti-UNG1/2 and anti-β-actin antibodies. In panel B, shRNA-transduced HeLa-CD4 cells were analyzed by indirect immunofluorescence using anti-UNG1/2 antibody (left part, right panels), and the number of shRNA-transduced cells without nuclear UNG2 staining was quantified over 100 cells (right part). Nuclei were stained with DAPI (left part, left panels). (C) Infectivity of viruses produced in shUNG2-transduced cells. Wild-type GFP reporter viruses were produced in shRNA-transduced 293T cells and then used to infect shRNA-transduced HeLa-CD4 cells as indicated. Viruses were normalized for CAp24 before infection. 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 293T cells and measured on shLuc-transduced HeLa-CD4 as target cells. (D) Interaction of Vpr with murine UNG2. 293T cells were transfected with plasmids for expression of GFP-tagged murine (mUNG2) or human (hUNG2) UNG2 in combination with the HA-tagged Vpr expression plasmid. Control (mock) corresponds to cells that did not express HA-Vpr. At 24 h after transfection, cell lysates (top and middle panels) were submitted to immunoprecipitation with anti-GFP antibody (bottom panel). Immunoprecipitates were analyzed by Western blotting with anti-GFP (top) and anti-HA (middle and bottom) antibodies. IP, immunoprecipitation. (E) Virion incorporation of mUNG2. shLuc- or shUNG2-transduced 293T cells were cotransfected with a wt HIV-1-based vector in combination with plasmids for expression of Flag-tagged mUNG2. Virions were collected from cell supernatants, and proteins from cell (top panel) and virion (middle and bottom panels) lysates were analyzed by Western blotting with anti-Flag (top and middle) and anti-CAp24 (bottom) antibodies. (F) Infectivity of viruses produced in shUNG2-transduced human cells expressing mUNG2. Wild-type GFP reporter viruses were produced in 293T shRNA-transduced cells expressing or not Flag-tagged mUNG2 and then used to infect HeLa-CD4 cells. Viruses were normalized for CAp24 before infection. 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 cells. Values are the means of at least four independent experiments. Error bars represent one SD from the mean. Statistical significance was determined by using the Student t test (n.s., P > 0.05; *, P < 0.05; **, P < 0.01).