TBK1 from orange-spotted grouper exerts antiviral activity against fish viruses and regulates interferon response

TANK-binding kinase-1 (TBK1) has been well studied in mammals because of its importance in type I interferon induction in antiviral immunity. However, the roles of fish TBK1 in virus infection still remained largely uncertain. In the current study, a TBK1 homolog from orange-spotted grouper (Epinephelus coioides) (EcTBK1) was cloned and its roles in fish viral infections were investigated. Sequence analysis showed that EcTBK1 encoded a 723-amino acid peptide which shared 98% and 73% identity to large yellow croaker (Larimichthys crocea) and human (homo sapiens), respectively. Multiple sequence alignments indicated that EcTBK1 contained conserved domains, including N-terminal kinase domain (KD), the middle ubiquitin-like domain (ULD) and C-terminal coiled-coil (CC) domains. The tissue distribution profiles demonstrated that EcTBK1 gene was constitutively expressed in all examined tissues, with predominant expression in intestine. Temporal expression analysis in vitro showed that the expression levels of EcTBK1 were significantly up-regulated in response to both red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection, suggested that EcTBK1 might exert crucial roles in fish virus infection. Subcellular localization indicated that EcTBK1 expression was primarily in the cytoplasm in GS cells. The ectopic expression of EcTBK1 significantly inhibited both SGIV and RGNNV replication. Furthermore, EcTBK1 overexpression significantly increased the expression levels of interferon related cytokines and pro-inflammatory factors. In addition, the overexpression of EcTBK1 increased the IRF3- and IRF7-regulated interferon promoter ISRE and IFN activity, and the regulatory effect on interferon immune response were dependent on its kinase domain. Together, we speculated that grouper TBK1 exerted antiviral activity against iridovirus and nodavirus via regulating the interferon immune and inflammatory response.