RIG-I and MDA5 are key cytosolic receptors for sensing various RNA viruses, including coronaviruses, influenza virus, and flaviviruses. Upon binding of viral RNA, RIG-I-like receptors initiate signal transduction pathways that result in type-I interferon (IFN) induction to suppress viral replication and the spread of infection. For their efficient replication, however, viruses have evolved elegant strategies to evade the IFN-mediated host immune response. Work from many laboratories has identified the precise mechanisms that lead to activation of RIG-I, which include RNA binding, K63-linked ubiquitination, oligomerization, and the re-distribution of RIG-I from the cytoplasm to mitochondria. In striking contrast, our knowledge about the mechanisms that govern activation of the related sensor MDA5 remains elusive. The proposed study builds on a recent discovery by the Gack laboratory that covalent modification of MDA5 by ISG15 activates MDA5 signaling by promoting its CARD-dependent oligomerization and cytosol-to-mitochondria translocation. Molecular and cell biological studies in ISG15-deficient human and mouse cells, including primary immune cells, demonstrated that ISG15 is required for an effective antiviral IFN response mediated by MDA5 (but not RIG-I). Our data also showed that MDA5 ISGylation is required for restricting the infection of SARS-CoV-2, Zika, dengue and picornaviruses. Furthermore, our most recent work revealed that SARS-CoV- 2 uses its papain-like protease (PLpro) to antagonize MDA5 by actively removing its ISGylation. Molecular, biochemical, and cell biological approaches combined with gene-targeting screens will focus on defining in precise detail how ISGylation regulates MDA5-mediated innate immunity, and also identify the E3 ligase that mediates MDA5 ISGylation and thereby its activation. We will also determine the relevance of MDA5 ISGylation for effective virus restriction using MDA5 gene-edited cells using CRISPR-Cas9 technology, and a novel mutant MDA5 knock-in mouse model. This study will also give detailed insight into the mechanisms by which SARS-CoV-2 and other coronaviruses counteract MDA5 ISGylation and determine the physiological role of this novel immune escape strategy in IFN antagonism, virus replication and pathogenesis. Our studies will provide a molecular understanding of the mechanisms that lead to activation of antiviral innate immunity, and also define a novel immune evasion strategy of coronaviruses, which may provide the foundation for new therapeutic approaches or help design better vaccines.