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 open reading frames (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 performed bulk RNA sequencing in MKL-2 cells, a Merkel cell carcinoma cell line, expressing MCPyV ALTO or RFP as a negative control via a doxycycline inducible lentiviral vector. The goal of the study was to identify Differentially Expressed Genes and pathways activated or inhibited via Gene Set Enrichment Analysis. MKL-2 cells were transduced with doxycycline inducible lentiviral vectors encoding Merkel cell polyomavirus ALTO or RFP as a negative control. Cells were selected with puromycin and then treated with doxycycline for 48 hours prior to harvest.