An increasing number of circular RNAs (circRNAs) have been found to be expressed by both viruses and human host cells during viral infection, yet the physiological significance of most circRNAs remains unclear. Our long-term goal is to use human polyomaviruses (HPyVs) to understand the regulation and function of circRNAs and to leverage this knowledge towards developing new diagnostics and treatments for viral diseases. We propose that many viral circRNAs function as coding RNAs that have distinct properties from linear RNAs spliced from the same gene. Specifically, our central hypothesis is that Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus encodes circular ALTO RNAs, circALTOs, that are translated to ALTO, which then modulates transcription in infected host cells. This model is based on preliminary studies showing that both MCPyV and TSPyV early regions efficiently generate circALTO, which are translated into ALTO protein in vitro. MCPyV circALTO functions as a transcriptional transactivator that can induce the transcription of multiple genes and pathways that have previously been implicated in DNA virus infections. We further demonstrate that a miRNA expressed from the MCPyV early region, miR-M1, negatively regulates circALTO and ALTO levels. Extending and validating this model would represent a ground-breaking advance in our understanding of circRNA function and regulation. This proposal will be organized into two aims: 1) Identify the factors that regulate the formation of MCPyV circALTO and its inhibition by the MCPyV miR-M1 miRNA and determine how these factors impact viral replication and persistence; 2) Determine how MCPyV ALTO modulates host cell transcription and whether ALTO’s effects on transcription are conserved in TSPyV. We will collaborate with virologists with expertise in polyomaviruses and miRNA biology to complete the proposed aims. Completion of this proposal will not only expand the relevance of circRNAs in virology and cell signaling but also significantly advance our understanding of the pathogenesis of human polyomaviruses.