Roles of RIG-1 N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-1 (RIG-1) recognizes viral RNA in a 5'-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDS of RIG-1 undergo the K-63-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-1 with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-1 first and second CARD in TRIM25-mediated RIG-1 ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T55I mutation in RIG-1 first CARD abolishes TRIM25 interaction, whereas the K172R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-1 function is further evidenced by a RIG-1 splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36-80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-1 multimerization and RIG-1-MAVS signaling complex formation, this SV effectively suppresses the RIG-1-mediated IFN-beta production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-1 activation but also identifies the RIG-1 SV as an off-switch regulator of its own signaling pathway.