TRIM25 phase separation drivers the RIG-I activation and restricted by influenza A virus NS1 to mediate immune evasion

ere we show that tripartite motif-containing protein 25 (TRIM25), a critical E3 ubiquitin ligase of RIG-I, undergoes LLPS in cells. And the intermolecular poly-hydrogen bond at 244-249 (-EY-EMK-) in the disordered region in coiled coil domain mediating its dimerization is important for TRIM25 phase separation. TRIM25 LLPS significantly weakened by amino acid mutations at 244-249 abolishes the activation of RIG-I K63 ubiquitination, type I interferon production and fails to inhibit influenza A virus (IAV) replication in human A549 cells. Furthermore, we found that initial TRIM25 at a liquid-like state is required for the formation of RIG-I signaling condensates and serves as microreactors to bind to dsRNA and recruits RIG-I, and subsequently catalyzes K63 ubiquitination of RIG-I to trigger downstream signaling activation. Intriguingly, IAV nonstructural protein 1 (NS1) undergoes LLPS under transfection or PR8-GFP virus infection states. NS1 protein was able to form condensates with TRIM25 more preferentially than RIG-I and led to the solidification of TRIM25, consequently blocks the formation of TRIM25 with RIG-I condensates and mediating immune evasion. Our results demonstrate that the TRIM25 LLPS drives the RIG-I signaling activation and also provides new perspectives for understanding mechanism for how viruses overcome innate immunity.