TRAF family member-associated NF-κB activator (TANK) regulates the antiviral function and NF-κB activation in red-spotted grouper (Epinephelus akaara)

Abstract

The TRAF family member-associated nuclear factor kappa B (NF-κB) activator (TANK) regulates the NF-κB activation through the TRAF-mediated signaling pathway and is involved in the antiviral pathway by inducing the interferon (IFN) production. In the present study, we identified a TANK ortholog from the red-spotted grouper (Epinephelus akaara) and analyzed its immunological functions. The coding sequence of EaTANK consists of 1047 base pairs and encodes a 348 amino acids protein. The predicted molecular weight and theoretical isoelectric point (pI) were 38.92 kDa and 5.39, respectively. According to the phylogenetic analysis, EaTANK was closely clustered with fish TANK orthologs, exhibiting the highest identity (97.1 %) and similarity (97.1 %) to that of Epinephelus lanceolatus. A highly conserved TBK1/IKKi binding domain (TBD) was identified between 110 and 164 residues. Our tissue distribution analysis showed that EaTANK mRNA was ubiquitously expressed in 12 tested tissues, with the highest expression in the spleen and peripheral blood cells (PBCs). According to the immune challenge experiments, EaTANK mRNA expression in PBCs was significantly elevated following stimulation with polyinosinic:polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS), or nervous necrosis virus (NNV). We also observed a significant elevation in the mRNA expression of downstream antiviral pathway-related genes (ISG15, IRF3, and IRF7) in EaTANK-overexpressing fathead minnow (FHM) cells against poly (I:C) stimulation. Moreover, the replication of 6 genes in the VHSV genome was inhibited by the overexpression of EaTANK. Finally, we confirmed that the expression of NFKB1 mRNA and promoter binding activity of NF-κB was significantly increased in poly (I:C)-stimulated EaTANK-overexpressing FHM cells. In conclusion, the results of this study suggest that TANK significantly contributes to the antiviral response and regulation of NF-κB activity in red-spotted grouper.