Identification of potassium and calcium channel inhibitors as modulators of polyomavirus endosomal trafficking

During virus entry, members of the Polyomaviridae transit the endolysosomal network en route to the endoplasmic reticulum (ER), from which degraded capsids escape into the cytoplasm and enter the nucleus. Emerging evidence suggests that viruses require both endosomal acidification and the correct ionic balance of K⁺ and Ca²⁺ ions in endosomes for correct virus trafficking and genome release. Here, using two polyomaviruses with different capsid architectures, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we describe methods to rapidly quantify virus infection using IncuCyte ZOOM imaging analysis, and use this system to investigate the role of both K⁺ and Ca²⁺ channels during the early stages of virus entry. Using broad spectrum blockers of both K⁺ and Ca²⁺ channels to specifically target host cell ion channel functionality, we show that MCPyV, but not SV40 can be inhibited by K⁺ channel modulators, whilst both viruses are restricted by the broad spectrum Ca²⁺ channel inhibitor verapamil. Using a panel of more specific Ca²⁺ blockers, we show that both MCPyV and SV40 are dependent on the activity of two-pore Ca²⁺ channels (TPCs), as the TPC-specific blocker tetrandrine prevented capsid disassembly and nuclear transport required for virus entry. We therefore reveal a novel target to restrict the entry of polyomaviruses, which given the known role of TPCs during endolysosomal-ER fusion, is likely to be applicable to other viruses that transit this pathway.