Amonkar, S. | Search bioRxiv

bioRxiv2025

E2F1-3 activate polyomavirus early transcription and viral replication

Merkel cell polyomavirus (MCPyV) is a DNA virus that establishes a persistent asymptomatic infection during childhood and can cause Merkel cell carcinoma (MCC) later in life. Its Large and Small Tumor antigens (LT, ST), splice variants of a common viral early transcript, drive viral replication and tumorigenesis by binding to and perturbing the function of host proteins. LT binds and inhibits RB1, deregulating E2F activity and host cell cycle control to permit viral replication during S phase. While the functions of LT and ST are relatively well characterized, how their expression is controlled is poorly defined. Here, we discovered that E2F1-3/DP1 dimers bind the MCPyV Non-Coding Control Region (NCCR) via a consensus E2 site close to the LT/ST transcriptional start site. Inhibiting E2F-NCCR binding, either by deleting the E2 site or treatment with a E2F small molecule inhibitor, downregulated LT/ST mRNA and protein expression in MCC cells and in 293A cells transfected with MCPyV. Our findings reveal an E2F/LT/RB1 positive feedback loop that appears to have evolved to support viral replication and is hijacked in MCC cells to promote cellular proliferation. Furthermore, we identified E2 sites in the NCCRs of PyVs closely related to MCPyV, including murine PyV, which mediate E2F/DP binding and potentiate viral early transcription. E2F/DP also bound weakly to the SV40 and BKPyV NCCRs, despite lacking an E2 site. Our findings challenge the prevailing model that PyV LT expression drives S phase entry and suggest, in contrast, that S phase entry stimulates PyV early transcription and replication. Significance statementPolyomaviruses (PyVs) express Large and Small Tumor antigens (LT, ST), splice variants of a common viral early transcript, that drive viral replication and tumorigenesis by binding and perturbing the function of host cells. LTs bind and inhibit RB1 via conserved LxCxE motifs, deregulating E2F activity and host cell cycle control to permit viral replication. Here, we discovered that E2F1-3 bind to the Non-Coding Control Regions (NCCRs) of many, but not all, PyVs to activate LT/ST transcription, revealing an E2F/LT/RB1 positive feedback loop that appears to have evolved to promote viral replication. Our findings challenge the existing paradigm that PyV LT expression drives S phase entry and suggest, in contrast, that S phase entry stimulates PyV LT/ST transcription and replication.

microbiology↗

bioRxiv2024

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or Alternate LT ORFs (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-{kappa}B signaling. ALTO activates NF-{kappa}B by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1).. Following activation, NF-{kappa}B dimers bind the MCPyV non-coding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-{kappa}B signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-{kappa}B and NTAR dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

microbiology↗