Fig 8.

Complementation of endogenous SMC1 with an SMC1 dominant negative mutant, hSMC1^{(S957A/S966A)}, rescues MVC infection-induced intra-S-phase arrest. (A) Immunofluorescence analysis of SMC1 phosphorylation. WRD cells were seeded on chamber slides 24 h prior to MVC infection. At 18 h p.i., cells were fixed and costained with anti-MVC NS1 and anti-p-SMC1(Ser957) antibodies and DAPI. Confocal images were taken at a magnification of ×100. Mock-infected cells were used as a negative control. (B to E) Analysis of SMC1 complementation with hSMC1^{(S957A/S966A)} on the cell cycle. WRD cells were transfected twice with siScrambled or siSMC1 siRNA and subsequently transfected with an empty vector, a plasmid expressing wild-type human SMC1A (hSMC1^wt) or dominant negative mutant hSMC1^{(S975A/S966A)}. (B) At 24 h posttransfection, cells were collected and analyzed by Western blotting using an anti-SMC1 antibody. The same membrane was reprobed for β-actin. The arrow shows a potential isoform of human SMC1. (C) At 24 h posttransfection, cells were infected with MVC or mock infected. Infected cells were incubated with BrdU for 1 h at 18 h p.i. Then cells were collected, treated with HCl, and costained with an anti-BrdU antibody and DAPI for cell cycle analysis by flow cytometry. Numbers shown in each histogram are percentages of the cell population in S phase and G₂/M phase, respectively, as indicated. (D) The statistical analysis of the percentage of cells in S phase, performed from three independent experiments, is shown. Data are shown as means ± standard deviations. P value was determined using Student's t test. (E) At 24 h posttransfection, mock- or MVC-infected cells were collected for Hirt DNA extraction and Southern blot analysis.