MDA5 associates with MAVS and its splicing variant in the antiviral response of large yellow croaker Larimichthys crocea.

As an essential intracellular sensor of the RLR family, MDA5 recognizes viral dsRNA and triggers host antiviral immune responses. In this study, we cloned and characterized the MDA5 homolog from large yellow croaker (Larimichthys crocea), named Lc-MDA5. The coding sequence of Lc-MDA5 is 2976 bp and encodes a 991 amino acids (aa) polypeptide, containing two N-terminal CARD motifs, a DEXDc, a HELICc, and a C-terminal RD domain. The gene consists of 16 exons and 15 introns. Tissue expression examination revealed widespread Lc-MDA5 expression in healthy fish, with maximal transcript levels in the liver, followed by the head kidney, gill, and spleen. Lc-MDA5 expression was strongly induced following stimulation with poly I:C, LPS, PGN, or Pseudomonas plecoglossicida. Confocal imaging showed that Lc-MDA5 exhibited cytoplasmic distribution. Co-immunoprecipitation and subcellular co-localization assays demonstrated that Lc-MDA5 interacts with either Lc-MAVS_tv1 or its transmembrane-deficient splice isoform Lc-MAVS_tv2 to form protein complexes. Overexpression of Lc-MDA5 activated the type I interferon (IFNd) promoter and upregulated IRF3 transcript levels. Moreover, SVCV replication was reduced, as indicated by decreased transcript levels of viral glycoprotein (SVCV-G) and matrix protein (SVCV-M). Co-expression of Lc-MDA5 with either Lc-MAVS_tv1 or Lc-MAVS_tv2 amplified promoter activities of IRF3, IRF7, and IFNd, elevated transcription of IRF3, IFNd, and ISG15, and co-expression with Lc-MAVS_tv2 further reduced SVCV-G and SVCV-M transcript levels relative to Lc-MDA5 or Lc-MAVS_tv2 alone. Overall, the present findings revealed that Lc-MDA5 contributes to host antiviral immunity by engaging MAVS and its splicing variant to activate downstream IRF3/7 and IFNd signaling cascades.