Grass carp DDX3X inhibits SVCV replication by activating IFN through the TRAF6-IRF3/7 signaling axis

Spring viremia of carp virus (SVCV) is one of the most devastating viral pathogens threatening the aquaculture of grass carp (Ctenopharyngodon idellus), and its outbreaks frequently result in severe economic losses worldwide. DEAD-box RNA helicase DDX3X has been established as a critical regulator of innate immune responses in mammals; however, its functional role and underlying mechanism in fish against SVCV infection remain largely unexplored. In this study, the coding sequence of grass carp DDX3X (gcDDX3X) was cloned and characterized. Sequence analysis revealed that gcDDX3X possesses conserved DEXDc and HELICc domains and shares high sequence similarity with DDX3X orthologs from other cyprinid fish species. Tissue expression profiling demonstrated that SVCV infection significantly upregulated gcDDX3X mRNA levels in multiple immune-related tissues, including the eye, intestine, liver, spleen, and gill. Consistently, poly(I:C) stimulation induced gcDDX3X expression in both GCO and EPC cells in a time- and dose-dependent manner. Subcellular localization analysis showed that gcDDX3X is predominantly distributed in the cytoplasm. Functional assays revealed that gcDDX3X overexpression markedly attenuated SVCV-induced cytopathic effects and plaque formation and significantly reduced viral titers, with more pronounced antiviral activity observed in GCO cells. Mechanistically, gcDDX3X overexpression substantially upregulated the transcriptional and protein levels of IRF3, IRF7, and type I interferon. Furthermore, co-immunoprecipitation and immunofluorescence co-localization assays demonstrated that gcDDX3X specifically interacts with grass carp TRAF6 (gcTRAF6) in the cytoplasm. Collectively, these findings provide the first evidence that grass carp DDX3X functions as a positive regulator of the IRF3/7-IFN signaling axis via TRAF6-mediated pathways to restrict SVCV replication. This study expands our understanding of the regulatory network of DDX3X in teleost innate immunity and offers a potential molecular target for developing therapeutic strategies against SVCV infection in grass carp aquaculture.