Single-cell analysis reveals host S phase drives large T antigen expression during BK polyomavirus infection.

BK polyomavirus (BKPyV) is a major cause of kidney transplant failure, for which there are no antivirals. The current model is that BKPyV expresses TAg (large T antigen) early during infection, promoting cells to enter S phase where the viral DNA can access the host replication machinery. Here, we performed a single-cell analysis of viral TAg expression throughout the cell cycle to reveal that robust TAg expression required replication of the host DNA first. By using inhibitors that only affect host and not viral replication, we show that both TAg expression and viral production rely on an initial S phase. BKPyV is known to promote cellular re-replication, where the cell re-enters S phase from G2 phase (without passing through mitosis or G1 phase) to prolong S phase for viral replication. Thus, BKPyV infection results in cells with greater than 4N DNA content. We found that these subsequent rounds of replication of the host DNA relied on canonical host cell cycle machinery and regulators despite BKPyV infection. Together, these findings suggest a model for polyomavirus replication, where robust viral TAg expression depends on an initial host S phase and that BKPyV primarily replicates during host re-replication. Having a better understanding of the molecular events that are required for BKPyV production will help identify effective therapeutic targets against BKPyV. Author summary: BK polyomavirus (BKPyV) is the leading cause of graft loss in kidney transplant patients due to the lack of effective antivirals. Previous studies have implied that early expression of a viral protein, large T antigen (TAg), promotes S phase entry as an essential first step in viral replication. Using single-cell analysis of BKPyV infected kidney cells, we show that viral protein expression relied on an initial host S phase since robust expression of TAg was found only in cells already in S phase. Additionally, blocking the initial host S phase prevented viral protein expression and decreased viral production. Once viral proteins were highly expressed, however, viral production no longer depended on host replication. Our findings support a new model where BKPyV initiates cell cycle entry independent of TAg since it was only expressed after an initial S phase. Under our new model, antiviral strategies could be developed to target BKPyV's dependence on the host cell cycle. Using these strategies will improve our understanding for how the virus replicates, enabling us to take a targeted approach for developing antivirals, and improve outcomes for kidney transplant recipients worldwide. [ABSTRACT FROM AUTHOR]