Functional characterization of duck TBK1 in IFN-β induction
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).
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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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