Decoding RIG-I ubiquitination in fish EPC Cells: Site identification and antiviral implications

Retinoic-acid-inducible gene-I (RIG-I)-like receptors (RLRs) comprise a family of DExD/H-box RNA helicases that are pivotal in antiviral and inflammatory responses. Ubiquitination serves as a crucial regulatory mechanism for both RIG-I activation and the type I interferon (IFN) signaling pathway in mammals. Although RLRs have been found to be evolutionarily conserved in teleost fish, the functional characterization of RIG-I ubiquitination in this vertebrate group remains largely unexplored. Through the integration of computational prediction with experimental validation, six ubiquitination sites (K115, K118, K145, K163, K168, and K171) were identified on RIG-I in Epithelioma papulosum cyprini (EPC) cells. Among these, K163, K168, and K171 are evolutionarily conserved in mammalian RIG-I orthologs. Biochemical analyses confirmed K63-linked ubiquitination at residues K115, K118, and K163. Functional characterization revealed that mutant RIG-I-K163R and RIG-I-K118R significantly downregulated ifn expression along with three interferon-stimulated genes (ISGs: gig1, mx1, and viperin) in EPC RIG-I knockout (EPC^rigi-/-) cells, demonstrating the essential role of these ubiquitination sites in RLR-mediated signaling activation. K118-mediated ubiquitination exerts a more pronounced regulatory effect on RIG-I activation compared to K163, as evidenced by enhanced spring viremia of carp virus (SVCV) proliferation in RIG-I-K118R-transfected EPC^rigi-/- cells relative to mutant RIG-I-K163R or wild-type controls. Notably, the mutant RIG-I-K115R exhibited enhanced antiviral activity, characterized by increased type I IFN signaling and reduced viral replication in EPC cells. This unexpected outcome may partially result from the mutant's increased self-oligomerization compared to wild-type RIG-I. It is suggested that a tunable regulatory mechanism mediated by multisite ubiquitination of RIG-I may be conserved in teleosts. These findings provide novel insights into the molecular mechanisms governing the RLR signaling pathway in teleosts, highlighting how multisite ubiquitination of RIG-I can lead to divergent antiviral outcomes.