BKV is a Polyomavirus that infects most of the human population. BKV typically establishes a lifelong, asymptomatic, persistent infection. Occasionally healthy humans excrete a small number of BKV particles in urine, and this, coupled with the fact that BKV grows productively in established kidney cell lines, has led to the view that the virus persists in the urinary tract. While harmless in most cases, BKV can undergo productive infection and induce an inflammatory response that results in serious diseases, including nephropathy and hemorrhagic cystitis in immunosuppressed patients. There is little knowledge as to why BKV is tropic for the kidney and whether its tropism is restricted to the urinary system. Furthermore, the factors governing the equilibrium between BKV productive infection and persistent infection are unknown. This application focuses on understanding the basis for cellular responses that lead to productive or persistent infection. BKV undergoes a productive infection in primary human renal proximal tubule epithelial cells (RPTE), characterized by extensive cell death and the release of about 40 infectious particles/cell. In contrast, BKV establishes a low-level persistent infection in vascular endothelial cells (VEC), causing minimal cytopathic effect with ~10% of the cells expressing viral proteins throughout two months of passaging. RNA-seq shows that BKV induces many cell cycle genes regulated by the E2F family of transcription factors in both RPTE and VEC. In addition, many interferon-stimulated genes (ISGs) are upregulated in response to BKV infection of VEC but not of RPTE. This research will use human primary human VEC cultures to identify the factors that influence infection outcomes: productive or persistent. Single cell transcriptomics will be applied to mock and BKV-infected VEC to assess the response of cell subpopulations to infection and to identify genes that play a role in restricting BKV replication. The activation of the interferon response by BKV infection in VEC will be assessed using both molecular and functional (CRISPR knockout) approaches to identify components of the pathway that are critical for limiting viral infection. Specific viral mutants will be used to determine what viral functions are required for the induction of the interferon response, as well as at which stage of the viral infection ISGs are induced. Click chemistry will be used to determine which cellular factors modulate the balance between productive and persistent infection. A combination of approaches will be used to identify cellular genes that distinguish restricted versus productively infected cells. This work will significantly advance our knowledge of BKV pathogenesis by characterizing the viral interaction with the innate immune system and by identifying cellular factors that promote or restrict BKV infection in a cell-type specific manner and will provide insights useful for the design of antiviral agents.
BK virus (BKV) causes serious diseases in transplant recipients and other patients with compromised immune systems. Successful infection requires that BKV redirects specific cellular systems towards viral replication, while simultaneously evades or neutralizes antiviral defenses. This proposal aims to identify cellular proteins and signaling pathways that differentially promote or restrict BKV infection in clinically relevant cell types.
