Pattern Recognition Receptors in the innate immune system serve as the first line of defense against pathogen infection. They recognize conserved molecular features commonly associated with pathogens and rapidly elicit anti-microbial immune response. One important family of such receptors are viral RNA receptors, RIG-I and MDA5, which cooperate with their common adaptor, MAVS, to activate the type I interferon response. The interaction between RIG-I/MDA5 and MAVS represents a committed step in initiation of the antiviral immune response and is often subject to multiple layers of regulation from both the host and invading viruses. Despite the importance, the molecular mechanism by which RIG-I and MDA5 interact with MAVS and link the upstream viral-detection events to the downstream signaling event is yet unclear. This is partly due to challenges of analyzing protein aggregation or oligomerization, which occurs during signal activation. We here propose to investigate the signal activation process of RIG-I, MDA5 and MAVS using an innovative "hybrid" approach that systematically integrates structural and biochemical analysis with cellular functional validation. In particular, we will focus on two key steps: (i) homo-oligomerization of te signaling domains (tandem caspase activation recruitment domain, 2CARD) of RIG-I and MDA5, which occurs upon their viral RNA recognition, and (ii) filament formation of MAVS CARD, which occurs upon its interaction with RIG-I/MDA5 2CARD oligomers. We will start with a model system consisting of the isolated signaling domains (i.e. 2CARD and CARD) to understand the detailed molecular and structural mechanisms for how RIG-I and MDA5 2CARDs oligomerize (Aim 1) and how the 2CARD oligomers trigger MAVS CARD filament formation (Aim 2). We will then investigate how the oligomerization and interactions among the signaling domains are regulated in the context of full-length RIG-I and MDA5 during viral RNA recognition (Aim 3). This proposal builds upon our novel findings, including filament formation of MDA5 and RIG-I (Peisley al, PNAS, 2010 & 2011; Mol Cell, 2013), the first crystal structure of the MDA5:dsRNA complex (Wu et al, Cell, 2013) and the recent, unpublished structures of the RIG-I 2CARD tetramer (in Aim 1A) and the MAVS CARD filament (in Aim 2A). These findings provide unprecedented opportunities to address key unresolved issues on the signal activation process of RIG-I and MDA5, both long-debated issues in the field and new questions arising from our discoveries. We expect that the proposed research would reveal novel molecular principles underlying the "assembly-mediated" signaling mechanism, an emerging paradigm for signal transduction in innate immunity and cell death. Furthermore, our mechanistic understanding could provide novel therapeutic strategies to harness the RIG-I/MDA5/MAVS pathways in treatment of immune disorders and development of antiviral or anticancer vaccine therapies.