Regulation of Host Innate Immunity Against Viral Infection

PROJECT SUMMARY: Innate immune sensors that detect viral pathogens act immediately upon infection by initiating signaling pathways that elicit the production of antiviral cytokines (such as type I interferons (IFNs}) or activate other cell-intrinsic host defenses. Furthermore, key molecules in innate immunity have repeatedly been shown to be major targets of viral antagonism. In the previous funding period of this R37 MERIT award, we addressed the fundamental mechanisms by which the host ISGylation machinery promotes the initiation of antiviral innate immunity, with a specific focus on the cytoplasmic RNA sensor MDA5. Moreover, we also investigated a novel evasion mechanism employed by SARS-CoV-2 and other coronaviruses to directly antagonize ISGylation-dependent MDA5 activation. Our work established that MDA5 requires ISGylalion to form higher-order oligomer assemblies and to initiate antiviral IFN responses, and further, revealed the in vivo relevance of MDA5 ISGylation. Moreover, our work showed that SARS-CoV-2 (and other coronaviruses) actively de-lSGylate MDA5 by utilizing their papain-like protease (PLpro) activity, which is part of the large Nsp3 protein. Our work identifying the molecular basis of MDA5 activation by ISG15 conjugation, and viral antagonism thereof, led us to ask the cell-type specific requirement of this novel regulatory mechanism of innate immunity. Moreover, (de}ISGylation events are known to regulate metabolic pathways which, in tum, may influence innate immune programs. Furthermore, Nsp3 is the largest protein encoded by the coronavirus genome and harbors, besides PLpro, several other domains which may also have immuno-modulatory activities. Therefore, in the extension period of this R37 MERIT award, we will use an integrative approach that combines molecular, biochemical, and cell biological techniques with cutting-edge proteomics and metabolomics screens as well as mouse infection studies to address fundamental questions in regulation of antiviral immunity by (de}ISGylation. We are conducting screens to identify novel (innate immune) substrates of coronaviral PLpro's de-lSGylating activity and will elucidate the mechanisms of how PLpro dysregulates host innate sensing pathways in different cell types, either through direct antagonism or by manipulating cellular metabolic pathways. We will also conduct mechanistic studies to identify PLpro-independent strategies of SARS-CoV-2 Nsp3 to dysregulate host immune signaling. Our studies will address fundamental unanswered questions in antiviral immunity, which may lay the groundwork for developing new antiviral therapeutic agents or help design better vaccines.