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. 2019 Jun 28;93(14):e00130-19. doi: 10.1128/JVI.00130-19

FIG 2.

FIG 2

Blocking mitosis prevented BKPyV-dependent host DNA damage when ATR was inactive. To determine if mitosis is required for DNA damage to occur during BKPyV infection when the DDR is inhibited, RPTE cells were transfected with siCdk1 or a control siRNA (siNTC) and then infected with BKPyV (multiplicity of infection of 0.5). At 24 hpi cells were treated with ATRi (5 μM VE-821), ATMi (10 μM KU-55933), or vehicle control (DMSO) and harvested at 72 hpi. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (A) Diagram of the G2/M checkpoint. DDR activation phosphorylates Cdk1 (inactive; gray) and arrests the cell cycle. In the absence of DDR activation, Cdk1 is active (green), and it complexes with cyclin B to promote mitosis. (B) To test if Cdk1 silencing blocked mitosis, mitotic cells were enumerated by FACS when the DDR was inhibited (ATMi or ATRi) with and without Cdk1 knockdown. Values are means ± standard deviations for n = 3 biological replicates. (C to E) Nuclear morphology was used to assess DNA damage to determine if Cdk1 silencing reduced DNA damage caused by DDRi. Quantification of fragmented nuclei (C) and quantification of nuclear morphology for >100 nuclei per biological replicate (n = 3) (D) are shown. Data from the experiment shown in Fig. 1B are shown again here for a direct comparison to siCdk1 nuclear morphology, which was determined as described for the experiments shown in Fig. 1B. Representative IFA with TAg (red) and DAPI (blue) is also shown (E). Scale bar, 100 μm. Values are the means ± standard deviations for n = 3 biological replicates. (F) DNA damage in BKPyV-infected (multiplicity of infection of 1.0) RPTE cells was quantified by alkaline comet assay. Comets from all biological replicates (n = 3) were combined. Error bars represent the 95% confidence intervals. Images are representative comets with various levels of DNA damage determined by the percentage of comet DNA in the tail. Scale bar, 50 μm. (G) Working model. Mitotic entry leads to DNA damage during BKPyV infection when ATR is inhibited.