Project Summary JCV and BKV are members of the Polyomavirus family that infect a large portion of the human population. Under normal conditions both viruses establish a lifelong, asymptomatic, persistent infection in the kidneys, and are frequently detected in urine and circulating blood without apparent consequences. While in most cases JCV and BKV are harmless, they can reactivate in immunosuppressed patients, and then induce serious diseases. However, the outcome from either infection is very different: BKV infection induces an inflammatory response and tissue damage in the kidney that results in nephropathy and hemorrhagic cystitis, while JCV reactivation causes fatal neurodegeneration in the brain, but only a few nephropathy cases. The factors governing the equilibrium between viral productive infection and persistent infection are unknown, as are the mechanisms controlling the different cellular responses to similar polyomaviruses. This application focuses on understanding the basis for cellular responses to JCV in a relevant primary human cell type, renal proximal tubule epithelial cells (RPTE). In contrast with BKV, whose infection induces extensive cell death around 5 dpi and the release of about 40 infectious particles/cell, JCV establishes a low-level infection in RPTE, causing minimal cytopathic effects and persisting for at least 3 weeks without inducing a full productive infection. Inoculation of RPTE with JCV results in induction of the interferon response, which could prevent viral expansion. This research will use human primary cells to identify the factors that influence infection outcomes: productive or persistent. Activation of the interferon response by JCV infection in RPTE will be assessed using both molecular and functional approaches to identify components of the pathway that are critical for responding to the virus and for limiting viral infection. Then, single cell transcriptomics will be applied to RPTE to assess the response of cell subpopulations to infection and to identify genes that play a role in restricting JCV replication. Finally, a comparison between the responses observed upon infection of RPTE with either BKV or JCV will be used to identify cellular genes that distinguish abortively versus productively infected cells. This application seeks to understand the unique responses to JCV in a clinically relevant human primary cell from the kidneys, the mechanisms that drive those responses and how the same cells respond in a different fashion to infection with BKV. This research will increase our understanding of principles governing cellular responses to viral infection, and how these responses influence the outcome of infection.