Summary EBV is a human tumor virus that is an etiological agent in Hodgkin's lymphoma, non-Hodgkin's lymphomas, stomach cancer and nasopharygeal carcinoma, and it is associated with an increased incidence of lymphomas in the HIV-infected patient context. EBV contributes to oncogenic progression through the expression of one or more viral genes that activate oncogenic pathways along the progression of stages that lead to cancer. A unique aspect of herpesviruses is their utilization of “latency” gene expression programs where no virus replication occurs and only a small subset of viral genes is expressed that remodels the host cell environment to facilitate viral maintenance and persistence. With some of the pathways altered by latency genes overlapping with pathway alterations required for oncogenesis, EBV latency genes are key contributors to the tumor phenotype. Because non-coding RNAs do not elicit adaptive immune responses, the utilization of viral non-coding RNAs in latency settings is a key herpesviral strategy to sustain viral persistence without promoting immune clearance. In cell culture models, we have discovered for the first time, that EBV expresses a class of largely non-coding RNAs referred to as circular RNAs (circRNAs) in both its latency and reactivation phases. The finding of a new repertoire of EBV transcripts is important because it stands to 1) add a previously unknown dimension to how EBV establishes successful infection and disease, 2) potentially provide novel and important mechanistic insights into EBV associated oncogenesis, and 3) provide new candidate therapeutic targets as well as liquid biopsy-based sentinel markers of disease. The goal of this R21 exploratory application is to perform vital validation experiments to determine whether these viral circRNAs are expressed in vivo in the natural tumor setting (aim 1) and to assess the possible relevance of this new class of viral RNAs in EBV infection and pathology through an initial investigation into the function of one of these (aim 2). Together, these experiments will provide key initial foundational work that will set the stage for initiating and prioritizing future investigations into the functions and mechanisms of action of this class of viral transcripts.