SUMMARY Effective immune defense against microbial infection depends upon efficient detection of pathogens by innate immune receptors. Among the proteins that ensure proper functioning of these immune receptors are ubiquitin (Ub) and E3 ligases that work through both proteasome-dependent and -independent mechanisms. In this grant, we explore the molecular mechanism of RIPLET, an E3 ligase that plays a proteasome-independent function in activating antiviral innate immune receptor, RIG-I. This grant builds upon our previous work on RIG- I and our recent findings on RIPLET. RIG-I is a conserved cytosolic innate immune receptor that recognizes RNAs from a broad range of viruses. RIG-I contains an N-terminal signaling domain (tandem CARD or 2CARD) and C-terminal RNA binding domain. Studies from our lab, and others, have identified at least three steps involved in the activation of RIG-I: (i) RNA binding, (ii) release of 2CARD auto-repression, and (iii) tetramerization of 2CARDs. The 2CARD tetramer then activates the downstream adaptor, MAVS, which in turn stimulates the antiviral signaling pathways. In particular, the third step of 2CARD tetramerization is stimulated by K63-linked polyubiquitin chains (K63-Ubn), which binds and stabilizes the 2CARD tetramer, as demonstrated by our crystal structures.! Despite the detailed understanding of the action of K63-Ubn on RIG-I, much remains debated about how and when K63-Ubn is placed on RIG-I. Accumulating evidence suggests that RIPLET, a poorly understood E3 ligase, plays an essential role in conjugating K63-Ubn required for 2CARD tetramerization. We found that RIPLET recognizes the RNA-binding domain of RIG-I, but only when it is pre-oligomerized on dsRNA in a filamentous form. We further revealed that RIPLET binds RIG-I filaments through two distinct binding modes: intra-filament binding and inter-filament bridging. The latter dominates for RIG-I filaments on longer dsRNAs, leading to RIG-I clustering and further amplification of RIG-I signaling in a dsRNA length-dependent manner. These findings showed the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination (Cadena et al, under revision, available in BioRxiv). These findings of ours now raise new and deeper questions about the RIG-I mechanism from the fresh perspective of RIPLET: precisely how RIG-I is ubiquitinated by RIPLET (Aim 1), how RIG-I is recognized by RIPLET (Aim 2), how the oligomeric state of RIG-I is altered by RIPLET (Aim 3), and whether RIPLET can be utilized to identify ligands for RIG-I (Aim 4). We here propose a combination of biochemistry, structural biology and cell biology to answer these questions, which we believe are the key to resolving the next layers of complexity in the RIG-I signaling pathway. The four aims will be pursued independently, but are highly synergistic. These four aims build upon our strong preliminary data, an established network of c...