Highlights d RIPLET, not TRIM25, is the obligatory ubiquitin E3 ligase for RIG-I d RIPLET recognizes pre-assembled RIG-I oligomers on dsRNA and ubiquitinates RIG-I d RIPLET can cross-bridge RIG-I filaments formed on longer dsRNA d The two binding modes synergize for length dependent dsRNA discrimination by RIG-I
The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner.These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self vs. non-self nucleic acid discrimination. local concentration of 2CARD (Peisley et al., 2013). Second, in addition to RIG-I filament formation, K63-linked polyubiquitin (K63-Ub n ) was also shown to promote RIG-I signaling (Jiang et al., 2012). Structural studies further showed that K63-Ub n chains bind the periphery of the core 2CARD tetramer, bridging the adjacent subunits and stabilizing its assembly . The 2CARD tetramer then acts as a helical template to nucleate the MAVS filament formation for downstream signal activation (Wu and Hur, 2015).Despite the detailed understanding of the action of K63-Ub n on RIG-I, much remains debated as to how and when Ub is placed on RIG-I, which E3 ligase is involved and how it interplays with RIG-I filament formation. Previous studies reported that TRIM25 and RIPLET (i.e. RNF135) are two essential E3 ligases important for RIG-I signaling (Gack et al., 2007;Gao et al., 2009;Oshiumi et al., 2009;Oshiumi et al., 2010). A more recent study proposed that RIPLET and TRIM25 sequentially act on RIG-I upon viral RNA engagement . That is, RIPLET first acts on the C-terminal portion of RIG-I, releasing 2CARD, which is then modified by TRIM25. However, given the previous finding that RNA binding is sufficient to release 2CARD (Kowalinski et al., 2011), it was unclear whether RIPLET indeed acts to release 2CARD and if so, how. At the same time, accumulating evidence suggested that TRIM25 has multiple, RIG-I-independent antiviral functions (Choudhury et al., 2014;Li et al., 2017;Manokaran et al., 2015;Meyerson et al., 2017;Zheng et al., 2017), raising the question whether the observed effect of TRIM25 on RIG-I represents a direct or an indirect effect.We here report a combination of cellular and biochemical data showing that RIG-I activation is dependent on RIPLET, not TRIM25, and that RIPLET suffices to ubiquitinate and activate RIG-I. In addition, RIPLET recognizes the filamentous form of RIG-I using two distinct binding modes, the interplays of which o...
Type I interferon (IFN) production by the proper activation of nucleic acid sensors is essential for hosts to eliminate invading viruses. Among these sensors, RIG-I-like receptors (RLRs) are well-known viral RNA sensors in the cytoplasm that recognize the nonself signatures of viral RNAs to trigger IFN responses. Recent accumulating evidence has clarified that some specific and atypical self-RNAs also cause activation of RLRs independently of virus infection. Importantly, when RLR-activation by these RNAs or a conformational change via missense mutations is sustained, the resulting continuous production of type I IFN will lead to autoimmune disorders. We, herein, focus on autoimmune diseases caused by chronic activation of RLRs and discuss possible mechanisms of their onset.
RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5′-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.
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