Identification and characterization of two DEAD-box RNA helicase DDX3 in largemouth bass (Micropterus salmoides) and their interaction with MAVS, TRIF, TBK1 and TRAF3.

DDX3, a member of the DExD/H-box protein family, is suggested to play an important role in antiviral immunity across mammals. However, its role in the fish immune response remains unclear. In this study, we isolated the cDNA sequence of DDX3a and DDX3b in largemouth bass (Micropterus salmoides), named MsDDX3a and MsDDX3b. The MsDDX3a cDNA contained an open reading frame (ORF) of 2103 bp and encoded a polypeptide of 700 amino acids. The MsDDX3b cDNA contained an ORF of 2259 bp and encoded a polypeptide of 752 amino acids. The predicted protein of MsDDX3a and MsDDX3b has two conserved domains, including the DEAD-like helicase superfamily DEXDc and C-terminal HELICc domains. In healthy largemouth bass, the MsDDX3a and MsDDX3b transcripts were broadly expressed in all examined tissues, with the highest expression in the liver and lowest expression in the stomach. After challenging with Nocardia seriolae and polyinosinic polycytidylic acid (Poly I:C), the expression level of MsDDX3a and MsDDX3b mRNA was upregulated or downregulated in all tested tissues. Overexpression of MsDDX3a and MsDDX3b in 293T cells showed a positive regulatory effect on NF-κB and IFN-β activity. Subcellular localization showed that MsDDX3a and MsDDX3b were expressed in the cytoplasm. Pulldown and coimmunoprecipitation (Co-IP) assays revealed that MsDDX3a did not interact with mitochondrial antiviral signalling protein (MAVS) and TIR domain-containing adaptor inducing interferon-β (TRIF). MsDDX3b did not interact with MAVS but did interact with TRIF. Moreover, MsDDX3a did not interact with TANK-binding kinase 1 (TBK1). In addition, both MsDDX3a and MsDDX3b can interact with tumour necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). These results provide a basis for further investigating the mechanism of MsDDX3 involvement in innate immune signalling. Overall, this study contributes to the elucidation of innate immune mechanisms in largemouth bass.