E2F1-3 activate polyomavirus early transcription and replication

Polyomaviruses (PyVs) are small, circular double-stranded DNA viruses that establish lifelong persistent infections. PyV infections are typically asymptomatic, but they can cause severe disease particularly in immunosuppressed individuals, including BKPyV-associated nephropathy, and Merkel cell carcinoma (MCC) caused by Merkel cell polyomavirus (MCPyV). While most individuals are infected with PyVs during childhood, it is unclear how these viruses persist in the host and what cellular signals prompt their reactivation. The early regions of PyV genomes encode regulatory proteins that control viral replication and host cell transformation in the case of MCPyV, SV40 and murine polyomavirus (MuPyV). All PyV early regions encode Large and Small Tumor antigens (LT, ST), which are splice variants of a common transcript. PyV LTs bind and inhibit RB1 via a conserved LxCxE motif, deregulating E2F transcriptional activity and cell cycle control to promote viral replication and tumorigenesis. The existing paradigm in the field is during normal PyV infection, LT is expressed and drives the host cell into S phase. However, a recent study revealed that cells infected with BKPyV only express LT and replicate the viral genome when already in S phase, but the mechanisms activating LT expression during S phase are unknown. The Non-Coding Control Region (NCCR), found in all PyVs, contains the viral origin of replication and predicted transcription factor (TF) binding sites, yet only a handful have been experimentally validated and shown to regulate PyV early transcription. Here, we set out to identify host TFs that bind the MCPyV NCCR to regulate early transcription in both MCC cells and in an in vitro model of MCPyV infection. We identified E2F1-3 as critical regulators of MCPyV early transcription, which bind to the MCPyV NCCR via a consensus E2 site just upstream of LT/ST transcriptional start site. We discovered E2 sites in the NCCRs of several other polyomaviruses, including MuPyV but not BKPyV. Deletion of the E2 sites in PyV NCCRs downregulates early transcription in luciferase assays. Despite not containing a consensus E2 site, we detected weak E2F binding to the NCCRs of BKPyV and SV40, suggesting that E2Fs also contribute to transcription of their early genes. Overall, our findings challenge the existing paradigm of PyV infection, that LT expression activates E2F signaling via RB1 inhibition, and suggest that E2Fs must already be active in the PyV-infected host cell for LT/ST expression and viral replication.