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

Description

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.

Steps to reproduce

FRAP experiments were performed on a Nikon NIS-Elements AI confocal microscope system. For cellular droplets, FRAP experiments were performed in a live cell imaging chamber at 37 °C. Human HEK293 or A549 cells were transfected with the pEGFP-TRIM25 plasmid for 12 h. Complete or partial photobleaching of the target droplets was achieved using a laser at 488 nm with about 10% laser power for 2 s. After bleaching, time-lapse images were obtained over a time course of 2-3 min. The fluorescence intensity of the region of interest (ROI) above was corrected by the unbleached control region and then normalized to the pre-bleached intensity of the ROI.

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