Activated Cdk1 is required for S-phase exit during BKPyV infection. To determine if the ATR pathway is activated during a BKPyV infection to prevent S-phase exit and mitotic entry of actively replicating cells, the contribution of Chk1i (2 μM; MK8776), ATRi (5 μM, VE-821), and Cdk1i (10 μM, RO-3306) to premature mitosis and cell cycle arrest were measured by FACS analysis of BKPyV-infected cells (multiplicity of infection of 0.5) at 72 hpi against a vehicle control (DMSO). (A) Cell cycle analysis was performed on Chk1i-treated cells (24 to 72 hpi) by FACS to identify S (EdU), G1, and G2 (based on DNA content staining) phases and M (pH3S10) phase. Representative contour plots (5%) are shown (top). The fractions of S-phase cells from the experiment shown in the top panel were graphed based on their DNA content and mitosis (pH3S10) to differentiate premature mitosis (red) from S phase (gray) (bottom). Data are representative of n = 3 biological replicates. (B) Diagram of Chk1i treatment during BKPyV infection. (C and D) The average percentages ± standard deviations of cells in S phase or premature mitosis were quantified for n = 3 biological replicates. Symbols are as follows: white, mock infection; black, BKPyV infection; square, vehicle; diamond, Chk1i. (E to G) Cdk1i was added to ATRi-treated, BKPyV-infected cells to determine if Cdk1 activity was required for ATRi to reduce S-phase levels. Briefly, mock-infected or infected RPTE cells were treated at 48 hpi with ATRi (5 μM, VE-821), Cdk1i (10 μM, RO-3306), or vehicle control (DMSO). Cells were pulse-labeled with EdU at 70 to 72 hpi to assess DNA synthesis and analyzed by FACS at 72 hpi for cell cycle distribution. Contour plots (5%) are representative of n = 3 biological replicates (E). The percentages of mitotic (pH3S10) (F) or S-phase (G) cells are presented as the means ± standard deviations for n = 3 biological repeats. Symbols are as follows: white, mock infection; black, BKPyV infection. (H and I) Cdk1i was used to synchronize BKPyV RPTE cells treated with ATRi, and then Cdk1 was washed out to determine if cells entered mitosis immediately (premature mitosis) or completed S phase prior to entering mitosis. Small-molecule inhibitor treatment and EdU labeling schema are shown for each ATRi treatment condition (control, Cdk1i, and Cdk1i wash) (H). The control, no-ATRi treatment, scheme is not shown. Cell cycle analysis (FACS) with (bottom) and without (top) ATRi was performed (I). Data shown are representative of n = 3 biological replicates. All events (mitosis, pH3S10-positive cells) were plotted as S phase (EdU+) versus DNA content. (J) Quantification for panel I, where Cdk1i was added (+) or not (−) or washed out (W) at 70 hpi, as diagrammed in panel H. The percentages of cells in mitosis (orange, positive for pH3S10) or premature mitosis (red, EdU+ and pH3S10+) were quantified as means ± standard deviations of n = 3 biological replicates. The fraction of premature mitosis is shown as a portion of overall mitosis in each group. Asterisks denote significant differences determined by one-way ANOVA between the overall mitosis population (black) and the premature mitosis population (red). (K) Working model. ATR prolongs S phase by activating Wee1 to inhibit mitotic entry by inhibiting Cdk1. Thus, ATR activation prevents premature mitosis, which in turn maintains S phase, prevents DNA damage, and enhances reduced viral titers. When ATR is inhibited, Wee1 is no longer activated to inhibit Cdk1. Activated Cdk1 in S phase induces premature mitosis, leading to DNA damage and reducing viral titers. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.