Regulation of RNA sensing and viral restriction by RNA structures

PROJECT SUMMARY Venezuelan equine encephalitis virus (VEEV) is a single-stranded positive-sense RNA virus transmitted by mosquitoes and is responsible for periodic epizootic/epidemic outbreaks of encephalitis in both horses and humans. The innate and interferon (IFN) responses are critical barriers for preventing the replication and spread of many viral pathogens including alphaviruses such as VEEV. As a result, viruses have evolved diverse mechanisms to inhibit or escape the innate immune response as well as antiviral effectors such as IFN-stimulated genes (ISGs). RNA structures are known to regulate basic viral processes (e.g. viral RNA transcription and translation), however, the role that viral RNA structure plays in shaping innate immune responses to viruses is understudied. We have previously shown that alphaviruses encode stable structures within their 5'-untranslated region (5'-UTR) that are critical for antagonizing IFIT1, an ISG important in restriction of non-self RNA. We have also shown that RNA structures in the 3'-UTR are important in modulating recognition of viral RNA by IFIT2. Recently we have shown that RNA structures in the 3'-UTR and E1 modulate replication of virulent and avirulent VEEV in macrophages, which are important targets of VEEV infection in vivo. The broad objectives of this proposal are to: 1) delineate E1 RNA structural determinants in virulent and avirulent VEEV that regulate macrophage replication, 2) define how E1 structural determinants recruit RBPs to the viral genome and the impact of this on host innate immune responses, 3) Define how macrophage replication fitness contributes to the differential pathogenesis in vivo of VEEV encoding virulent and avirulent RNA structures, and 4) Define how macrophage replication fitness shapes innate immune responses in vivo. Findings from these studies will provide key insight into novel mechanisms of VEEV pathogenesis and emergence of pathogenic variants. This will have broad implications for our understanding of viral emergence and development of RNA-based therapeutics.