Elsevier

Cytokine

Volume 111, November 2018, Pages 325-333
Cytokine

Functional characterization of duck TBK1 in IFN-β induction

https://doi.org/10.1016/j.cyto.2018.09.007Get rights and content

Highlights

  • duTBK1 is ubiquitously expressed in various duck tissues.

  • duTBK1 contributes to the type I IFN induction via the activation of IRF1 and NF-κB.

  • The kinase domain and ubiquitin-like domain of duTBK1 are essential for IFN-β induction.

  • Overexpression of duTBK1 reduced the replication of DRV and DTMUV.

Abstract

TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) serves as hub molecule at the crossroad of multiple signaling pathways of type I interferon (IFN) induction. The importance of TBK1 in innate immunity has been demonstrated in mammalian, however the characterization and function of TBK1 in avian remains largely unknown. In this study, we cloned duck TBK1 (duTBK1) from duck embryo fibroblasts (DEFs) for the first time, which encoded 729 amino acids and had a high amino acid identity with goose and cormorant TBK1s. The duTBK1 showed a diffuse cytoplasmic localization in DEFs and was extensively expressed in all tested tissues. Overexpression of duTBK1 induced IFN-β production through the activation of IRF1 and NF-κB in DEFs. The N-terminal kinase domain and the ubiquitin-like domain in middle of duTBK1 played pivotal roles in IFN-β induction as well as in IRF1 and NF-κB activation. Furthermore, knockdown of duTBK1 by small interfering RNA significantly decreased poly(I:C)- or Sendai virus (SeV)-induced IFN-β expression. In addition, duTBK1 expression dramatically reduced the replication of both duck reovirus (DRV) and duck Tembusu virus (DTMUV) in DEFs. These results suggested that the duTBK1 played a pivotal role in mediating duck antiviral innate immunity.

Introduction

The trigger of antiviral innate immune response relies on the recognition of pathogen-associated molecular patterns (PAMPs) derived from viruses by specialized receptors called molecular pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) [1], [2]. PRRs-induced signal transduction pathways are activated by detecting the PAMPs, including viral DNA or RNA, transcription products, and replicative intermediates [3]. Upon recognition of PAMPs by plasma membrane-localized or cytosolic PRRs, different kinds of adaptor proteins, such as TRIF, MAVS, MyD88, and STING, are recruited by different PRRs to initiate the downstream induction of type I IFN and inflammatory cytokines via activation of NF-κB, IRF3 and AP-1, mediating the effective and further elimination of invading viruses [4], [5], [6].

TBK1, also known as NF-κB-activating kinase (NAK) or TRAF2-associated kinase (T2K), is a member of non-canonical IκB kinase (IKK) family and can directly phosphorylate IKKβ, thus activating NF-κB through inducing the degradation of IκB in human, mouse [7], [8]. Furthermore, TBK1 has also been reported to assemble with TRAF3 and TANK, upstream adaptor proteins of TBK1, to phosphorylate IRF3, leading to the nucleus translocation of IRF3 and inducing the expression of type I IFN [9], [10]. Mammalian TBK1 has been described as a protein of 729 amino acids consisting of a kinase domain (KD) at N-terminal, a middle ubiquitin-like domain (ULD) which controls the activity of kinase domain, and a coiled coil-containing domain (CC) in the C-terminal of unknown function [11], [12]. The KD and ULD domains are both essential for the phosphorylation of downstream IRF3 and NF-κB, thus triggering the IFN production [13], [14].

Although the characterization and function of TBK1 in antiviral immunity has been identified in mammals, the characterization of TBK1 gene and its roles in avian innate immunity remain largely unknown. Recently, chicken TBK1 has been reported to contribute to IFNβ induction against chicken avian leukosis virus subgroup J (ALV-J) infection [15]. In the present study, a full-length duck TBK1 (duTBK1) gene was characterized for the first time. Moreover, we revealed that the duTBK1 played an important role in inducing duck IFN-β expression and possessed the ability to dramatically inhibit proliferation of both ducks reovirus (DRV) and duck Tembusu virus (DTMUV).

Section snippets

Cells, tissues, viruses, and reagents

Duck embryo fibroblast cell line was obtained from ATCC and cultured in Minimum Essential Medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). Tissues for TBK1 expression analysis obtained from 1-month-old healthy cherry ducks, including lung, liver, spleen, heart, kidney, cerebrum, thymus, duodenum, caecum and bursa of Fabricius were snap-frozen into liquid nitrogen and stored at −80 °C for further analysis. The NF-κB specific inhibitor BAY11-7082 was

Cloning and sequence analysis of duTBK1

The rapid amplification of cDNA ends (RACE) PCR was used to characterize duTBK1, and specific primers were designed basing on the predicted Anas platyrhynchos duTBK1 coding sequence (XM_005029450) (Fig. 1A). As a result, the full-length cDNA sequence of duTBK1, which is 3794 bp in length, was obtained and uploaded to GeneBank (accession number MG772817). Based on the information provided by the National Center for Biotechnology Information (NCBI), duTBK1 gene was proved to locate on an Unplaced

Discussion

TBK1 has been demonstrated as a regulator of IFN-β expression via IRF3/7 or NF-κB signaling pathways, thus mediating the innate immunity in human [8], [9]. Moreover, mouse and fish TBK1 also triggers the IFN-β induction to combat the infection of Sev and grass carp reovirus (GCRV), respectively [7], [10], [21]. However, the characterization and function of TBK1 in duck remain largely unknown. Here, we cloned and characterized the duTBK1 gene for the first time to expand knowledge of avian

Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (31772737), Hubei Province Natural Science Foundation for Innovative Research Groups (2016CFA015), Applied Basic Research Project of Wuhan (Grant No. 2017020201010227) and the Fundamental Research Funds for the Central Universities (grant number 2662017PY066).

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