Primers used for cloning and qRT-pCR

Figure S1. Protein sequence alignment of HPyV6, HPyV7 and MCPyV small T antigens. Figure S2. Expression of HPyV sT induces p53-dependent senescence in human fibroblasts. Figure S3. Proliferation of BJ fibroblasts with expression of HPyV sT. Figure S4. Analysis of RNA-Seq from HPyV6, HPyV7, and MCPyV sT expressing BJ fibroblasts. Figure S5. Time course and extended analysis of additional SASP gene expression in BJ cells after sT expression. Figure S6. MCPyV sT shRNA confirms role of sT in SASP gene expression. Figure S7. Impact of LSD motif of MCPyV sT on gene expression and proliferation. Figure S8. MCPyV st, but not HPyV6/7 sT, stabilizes and activates c-Myc in an LSD motif dependent manner. Figure S9. Chromatin remodeling signatures induced by MCPyV sT. Figure S10. Mechanism of ncNF-κB and SASP induction by MCPyV sT. Figure S11. ncNF-κB signaling is required for SASP and EZH2 expression and affects cell proliferation. Figure S12. PyV sT conditioned media is not sufficient for growth in low serum. Figure S13. ST expression in VP-MCC lines and tumors. Figure S14. Impact of ncNF-κB inhibition on VP-MCC and VN-MCC. Figure S15. Summary Figure.

Circular RNAs (circRNAs) are an emerging class of RNAs with diverse functions. We previously described human papillomavirus derived circRNAs with transforming activity. Here, we describe circular RNAs encoded by Merkel Cell Polyomavirus (MCPyV), including two that are circular forms of the previously described alternative open reading frame (ALTO) gene in the early region of MCPyV (circALTO1 and circALTO2). CircALTOs can readily be detected MCPyV-positive Merkel Cell Carcinoma (MCC) by both inverse PCR and northern blot. Both circALTOs contain the ALTO open reading frame and are able to ALTO peptides. CircALTOs are stable, predominantly located in the cytoplasm, and modified with N6-methyladenosine (m6A). Thus, we describe the first known polyomavirus-encoded circRNAs (circALTO1/2), which encode for proteins, and may contribute to both MCPyV replication and MCC tumorigenesis.

Abstract Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB signaling prevented VP-MCC cell growth in vitro and in xenografts. We identify MCPyV sT–induced ncNF-κB signaling as an essential tumorigenic pathway in MCC. Implications: This work is the first to identify the activation of ncNF-κB signaling by any polyomavirus and its critical role in MCC tumorigenesis.

Several polyomaviruses, including Human Polyomavirus 6 (HPyV6) and Human Polyomavirus 7 (HPyV7), are shed chronically from human skin. HPyV6 has been detected in the lymph node of a patient with Angiolymphoid Hyperplasia with Eosinophilia (ALHE), and HPyV7 has been strongly linked to a pruritic skin eruption with a characteristic histology in two immunosuppressed transplant recipients. We screened skin biopsies—14 cases of ALHE and 6 cases of pruritic eruptions showing "peacock plumage" on histology—by PCR for human polyomaviruses. Low levels of HPyV6 or 7 DNA were detected in several cases of ALHE, but neither virus could be detected by immunohistochemistry (IHC). Interestingly, several pruritic and dyskeratotic dermatoses were strongly positive for HPyV 6 or 7 by PCR. These cases were further analyzed by IHC, electron microscopy (EM), immunofluorescence, quantitative PCR, and sequencing. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 infected keratinocytes. High viral copy number in lesional skin, the expression of viral proteins by IHC in dyskeratotic keratinocytes, and the identification of intact virions by EM and sequencing support a role for active viral infections in these skin diseases. In conclusion, we report that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a unique histologic pattern and describe these entities as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses (H6PD and H7PD)."