MDA5 plays a critical role in antiviral innate immunity by functioning as a cytoplasmic double-stranded RNA sensor that can activate type I interferon signaling pathways, but the mechanism for the activation of MDA5 is poorly understood. Here, we show that TRIM65 specifically interacts with MDA5 and promotes K63-linked ubiquitination of MDA5 at lysine 743, which is critical for MDA5 oligomerization and activation. Trim65 deficiency abolishes MDA5 agonist or encephalomyocarditis virus (EMCV)–induced interferon regulatory factor 3 (IRF3) activation and type I interferon production but has no effect on retinoic acid–inducible I (RIG-I), Toll-like receptor 3 (TLR3), or cyclic GMP-AMP synthase signaling pathways. Importantly, Trim65−/− mice are more susceptible to EMCV infection than controls and cannot produce type I interferon in vivo. Collectively, our results identify TRIM65 as an essential component for the MDA5 signaling pathway and provide physiological evidence showing that ubiquitination is important for MDA5 oligomerization and activation.

Aberrant activation of the NLRP3 inflammasome contributes to the evolution of diverse inflammatory diseases. Inhibition of the NLRP3 inflammasome has been proven to be an effective treatment strategy for NLRP3‐driven diseases. This study revealed that multiple natural diterpenes from Isodon plants can inhibit the NLRP3 inflammasome, among which Rosthornin B (Ros B) exhibited the best inhibitory effect, with an IC ₅₀ of 0.39 μM. Further study revealed that Ros B directly interacts with NLRP3, thereby restraining NEK7‐NLRP3 interaction and inhibiting NLRP3 inflammasome assembly and activation. Remarkably, Ros B had a significant therapeutic benefit in mouse models of NLRP3‐driven septic shock, peritonitis, and colitis. Our study has identified a series of natural diterpenes that target the NLRP3 inflammasome. These natural diterpenes, especially those with low IC ₅₀ values, may lead to the development of new drugs and potential clinical therapies for diseases driven by NLRP3 inflammasome activation.