The early detection of invading viruses by the host depends on a limited number of intracellular receptors that sense conserved structural components of viruses and subsequently initiate signaling cascades leading to IFN-1/2 induction. Retinoic acid-inducible gene I (RIG-I) has emerged as a key cytosolic receptor for sensing RNA viruses, including influenza virus and hepatitis C virus. In addition, tripartite motif (TRIM) proteins, containing a RING domain with potential ubiquitin E3 ligase activity, represent a novel class of anti-viral molecules involved in innate immunity. On the other hand, many viruses have evolved sophisticated mechanisms to counteract host protective IFN-mediated anti-viral pathways. Despite the recent rapid progress in deciphering molecular components in the RIG-I signaling pathway, the regulation of its anti-viral activity has not been well studied. However, it is exactly the regulation of the host IFN production, which dictates the outcome of the viral infection and pathogenesis. Our preliminary studies have demonstrated that the interconnection between the cytosolic sensor RIG-I and a member of the TRIM family creates a potent intracellular host defense mechanism required for type I IFN induction upon viral infection. RIG-I undergoes robust Lys 63- linked ubiquitination induced by TRIM25, enabling RIG-I to interact with the CARD-containing adaptor MAVS, and thereby triggering anti-viral signal transduction to limit viral replication. The proposed study is directed toward investigating how the host innate immune system recognizes virus infection and triggers IFN responses, with a specific focus on RIG-I and TRIM25 proteins. Biochemical, cell biological and structural studies will focus on defining how posttranslational modifications affect the RIG-I signaling activity to limit viral replication (Aim 1). This study will further detail the mechanisms by which influenza A virus NS1 counteracts the RIG-I/TRIM25 signaling pathway and its role for the pathogenesis of influenza virus infection in vivo (Aim 2). Insights gained from this study will not only illuminate new views on the regulatory networks of innate immunity, but also delineate the molecular mechanisms underlying host-viral interaction, thereby providing foundations for developing novel therapeutic strategies for emerging virus- associated disorders.