A Two-Site Interaction Underpins TRIM25 Activation of the RIG-I Anti-Viral Response

SPRY/B30.2 domain-containing proteins are found in 103 human proteins that regulate numerous cellular processes including RNA processing, histone methylation, red blood cell membrane integrity, terminal erythroid cell differentiation and innate immune responses. Despite an increased understanding of SPRY/B30.2 domain function, some confusion remains as to the precise domain boundaries, the number and location of binding sites, and importantly how activation of these domains contribute to signal transduction. The B30.2 domain of tripartite motif 25 (TRIM25) interacts with the viral RNA sensor retinoic acid-inducible gene I (RIG-I), facilitating the formation of a RIG-I tetramer, which is required for activation of the innate immune response against RNA viruses such as influenza A, measles, HIV and hepatitis C. Here we have investigated the biochemical and structural changes associated with the activation of TRIM25 in the context of RIG-I binding. We solved a 1.8 Å crystal structure of the TRIM25 B30.2 domain and identified a putative RIG-I-binding site, which was confirmed by mutagenesis and functional analyses. Further mutagenesis identified a second site on the opposing face of the B30.2 domain that also specifically interacts with RIG-I. While the CARD1 domain of RIG-I is known to interact with the B30.2 domain of TRIM25, our data suggest that a conformational change within the CARD2 domain of RIG-I enables it to also interact with the B30.2 domain of TRIM25. We propose that both RIG-I CARDs interact with sites on opposing sides of the TRIM25 B30.2 domain to form a TRIM25/RIG-I complex that facilitates RIG-I tetramerisation and activation. The characterization of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family. Disease-associated polymorphisms of Pyrin, involved in Familial Mediterranean Fever, and mutational analyses of TRIM5a, required for HIV restriction, suggest that a second binding site may be a characteristic feature of other B30.2-containing TRIM proteins, and may facilitate the formation of large oligomeric complexes that are important for inflammasome function and HIV capsid destruction.