Fig 9.

MVC infection-induced DDR and intra-S-phase arrest are dependent on replicating viral DNA. (A) Comet assay analysis. WRD cells were mock or MVC infected. At 18 h p.i., half of the cells were collected and analyzed for cells with damaged DNA (Comet+) by Comet assay; the other half of the cells were fixed and costained with an anti-NS1 antibody and DAPI to quantify the percentage of infected (NS1+) cells by immunofluorescence analysis. Confocal images were taken at a magnification of ×40. A statistical analysis of the percentage of cells with damaged DNA was performed from three independent Comet assays. Data are shown as means ± standard deviations. (B) Flow cytometry analysis of the cell cycle in transfected cells. WRD cells were transfected with the indicated plasmids for 18 h and then were incubated with BrdU for 1 h. The cells were collected, treated with HCl and costained with DAPI, anti-BrdU and anti-NS1 antibodies (for pIMVC and mutant derivatives), or an anti-Flag antibody (for pLenti-based plasmids) for flow cytometry analysis. (C and D) The MRN complex is associated with replicating viral DNA. Mock- or MVC-infected cells were incubated with BrdU for 1 h at 18 h p.i. (C) Immunofluorescence analysis of the MRN complex. The cells were fixed and costained with the indicated antibodies and DAPI. Confocal images were taken at a magnification of ×100. (D) Coimmunoprecipitation (Co-IP) with an anti-BrdU antibody. The BrdU-labeled cells were lysed and centrifuged. The supernatant containing BrdU-labeled viral ssDNA was immunoprecipitated with an anti-BrdU antibody. Immunoprecipitated samples were blotted with an anti-Mre11 antibody. An amount of lysate equal to 5% was used as an input control for each sample. Arrows show the Mre11 bands, whereas arrowheads show the immunoprecipitated light and heavy chains of the IgG antibody.