Identity and Accessibility of Immunostimulatory Viral RNAs During Flavivirus Infection

Project Summary This project aims to reveal the molecular mechanisms underpinning initiation of innate immune activation and the innate immune response during flavivirus infection. Flaviviruses comprise a number of emerging and re- emerging pathogens with a global disease burden of over 400 million infections each year. West Nile virus (WNV) is an emerging neurotropic flavivirus that serves as a model flavivirus and is itself the number one arbovirus infection in the United States. Infection with WNV elicits a primary innate immune response through activation of the RIG-I-like receptor pattern recognition receptor signaling pathway when RIG-I detects viral pathogen associated molecular patterns (PAMP). However, the identity and features of viral RNA (vRNA) that drive this response as well as how sequestered vRNA is sensed by cytosolic RIG-I remain unknown. The proposed research aims to identify the species of and molecular motifs within WNV vRNA that drive a productive innate immune activation and response (Aim 1) and to pinpoint the intracellular sites of vRNA-RLR interaction in the infected cell to define the relationship between vRNA localization within/outside of viral replication compartments and innate immune activation (Aim 2). Our preliminary data has uncovered a role for single-stranded viral replication intermediates (negative-sense vRNA) in driving early innate immune activation. In Aim 1, cellular subpopulations will be sorted based on innate immune activation status then subjected to RNA sequencing to further identify specific species of vRNA associated with innate immune activation. Further analysis of vRNA bound to RIG-I during infection will be performed with immunoprecipitation followed by RNA sequencing and specific immunostimulatory nucleotides will be mapped via individual nucleotide resolution cross-linked immunoprecipitation (iCLIP). In Aim 2, novel vRNA-RLR proximity ligation assays alongside immunofluorescence will be used to pinpoint cellular locations of innate immune activation. Innovative cryo- electron tomography (cryo-ET) coupled with RNA fluorescent in situ hybridization via correlative light and electron microscopy (CLEM) will be used to define the relationship between immunostimulatory RNAs and viral replication compartments at the ultrastructural level. In addition to revealing the identify and location of immunostimulatory vRNAs during WNV infection, the work in this proposal will facilitate training in multiple cutting-edge techniques including iCLIP, RNA sequencing and bioinformatics, and high-resolution imaging including cryo-ET and CLEM, with the support of co-sponsors who are experts in flavivirology and innate immunity and viral ultrastructure and imaging techniques. Technical and conceptual training provided will support the transition of the trainee to an independent investigator in RNA virology and innate immune activation, while research findings will provide conceptual advances in our understanding of RLR-signaling during flavivirus infection.