Functional Characterization of Dark Sleeper (Odontobutis obscura) TBK1 on IFN Regulation.

Jian Chen,Shun Li,Long Feng Lu,Xi-Yin Li,Pei Li,Zhuo Cong Li

doi:10.3389/fimmu.2019.00985

Abstract

In East Asia, the dark sleeper, Odontobutis obscura (O. obscura) is a crucial commercial species of freshwater fish; however, its molecular biology research is still undeveloped, including its innate immune system, which is pivotal to antiviral responses. In this study, we cloned and identified the characterization and kinase function of dark sleeper TANK-binding kinase 1 (TBK1), supplementing the evidence of the conservation of this classical factor in fish. First, the ORF of Odontobutis obscurus (O. obscura) TBK1 (OdTBK1) was cloned from liver tissue by RACE-PCR. Subsequent nucleic acid and amino acid sequence analysis suggested that OdTBK1 is homologous with other fish TBK1, and the N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD) are conserved. Subsequently, the cellular distribution demonstrated that OdTBK1 was located in the cytoplasm region. With regard to the identification of functions, OdTBK1 activated several interferon (IFN) promoters' activity and induced downstream IFN-stimulated genes (ISGs) expression. In a canonical manner, wild-type OdTBK1 significantly phosphorylated interferon regulatory factor 3 (IRF3) but failed when the N-terminal region was truncated. Furthermore, overexpression of OdTBK1 decreased viral proliferation remarkably. Collectively, these data systematically analyzed the characterization and function of OdTBK1, initiating the study of the innate antiviral response of dark sleeper.

Highlights

A variety of pattern recognition receptors (PRRs) mediate a host’s innate immune response to pathogen invasion; six families have been identified, including Toll-like receptors (TLRs), C-type lectins (CTLs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), AIM2-like receptors (ALRs), and OAS-like receptors (OLRs)
The coding sequence (CDS) of OdTBK1 was obtained by RACEPCR
To investigate the homology of OdTBK1, phylogenetic analyses of TANK-binding kinase 1 (TBK1) with several species were conducted by MEGA7 based on the CDS sequences; these members could be divided into mammals, amphibians, mollusks, and fish (Figure 1)

Summary

Introduction

A variety of pattern recognition receptors (PRRs) mediate a host’s innate immune response to pathogen invasion; six families have been identified, including Toll-like receptors (TLRs), C-type lectins (CTLs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), AIM2-like receptors (ALRs), and OAS-like receptors (OLRs). RLRs are cytoplasmic sensors of RNA [1] Upon activation, they signal to the mitochondrial antiviral signaling protein IPS-1, Cardif, or VISA) [2,3,4] to form active MAVS polymers and subsequently recruit tumor necrosis factor (TNF) receptorassociated factor (TRAF) family ubiquitin E3 ligases to synthesize polyubiquitin chains, which activates IKK and TANK-binding kinase 1 (TBK1). TBK1 is a serine/threonine protein kinase of the IKK kinase family involved in innate immunity to viral infection by inducing type I IFNs and mediating TANK’s ability to activate NF-κB [6, 8]

Methods

Results

Conclusion