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Irf3 from mandarin fish thymus initiates interferon transcription

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Abstract

Interferon regulatory factors (IRFs) are transcription factors of the interferon (IFN)-inducible signaling pathway essential for host immunity against antimicrobial infection by virus and bacteria. Interferon regulatory factor 3 (IRF3) regulates the expression of IFNs and IFN-stimulated genes by binding to the IFN stimulatory response element (ISRE). In this study, we analyze the thymus transcriptome of the mandarin fish Siniperca chuatsi and report the functional analysis of Irf3 from the thymus as an emerging model of antiviral approaches. The predicted S. chuatsi IRF3 (Sc-Irf3) protein has 465 amino acid residues and evolutionarily conserved domains and is clustered in the IRF3 subfamily on a phylogenetic tree. Sc-Irf3 upon transgenic expression was mainly found in the cytoplasm through Western blot analysis and microscopy, but it translocated to the nucleus after polyinosinic:polycytidylic acid (ploly I:C) treatment. Endogenous Sc-irf3 RNA expression was detected in all eight adult organs examined. Importantly, Sc-irf3 RNA expression was significantly upregulated by ploly(I:C) treatment in the adult organs. Concurrently, reporter assays revealed that Sc-Irf3 increased the transcriptional activity of the ifnβ promoter, a minimal ISRE-containing promoter, and ifn promoter of mandarin fish. Therefore, Sc-Irf3 plays a major role in the IFN immune defense system against virus infection.

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Funding

This work was financially supported by the China Agriculture Research System (CARS-46), the Shanghai Collaborate Innovation Center for Aquatic Animal Genetics and Breeding (ZF1206), and the Talent Development Special Fund of Anhui Academy of Agricultural Sciences (16F0505).

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Authors and Affiliations

  1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China

    Xiaowu Chen, Yawei Shen & Jinliang Zhao

  2. Fisheries Research Institute, Anhui Academy of Agricultural Sciences, NO.40 South Nongke Road, Luyang District, Hefei, 230000, Anhui, China

    Minglin Wu

  3. Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China

    Jinliang Zhao

  4. Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai, 201306, China

    Jinliang Zhao

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  1. Xiaowu Chen
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  2. Yawei Shen
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Correspondence to Jinliang Zhao.

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All animal experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved and authorized by the State Council of the People’s Republic of China.

Electronic supplementary material

Figure S1

cDNA sequence of SC-irf3 and the 5′-flanking sequence of SC-ifn. (A) cDNA (black letters) and deduced protein (blue letters) sequence of SC-irf3; (B) Sequence of the 5′-flanking region of Sc-ifn. Boxed ATG indicates the start codon site. The predicted TATA box is boxed. Predicted NF-kappaB binding sites are wave lined, and the interferon-stimulated response element (ISRE) is underlined. (PNG 6647 kb)

High resolution image (TIF 10288 kb)

Figure S2

Multiple alignment of Sc-Irf3 with its homologues. Residues shaded in black are completely conserved across all species, and residues shaded in gray are similar in terms of side chains. The dashes in the amino acid sequences indicate the gaps introduced to maximize alignment. Putative DNA-binding domain (DBD) in the N-terminal and IRF association domain (IAD) in the C-terminal are underlined. Serine-rich C-terminal domain is boxed. Black triangle represents the tryptophan residue cluster. (PNG 7481 kb)

High resolution image (TIF 12587 kb)

Figure S3

Phylogenetic analysis of the IRF3 family phylogenetic tree was constructed with the neighbor-joining method by Mega 6.0. Node values represent percent bootstrap confidence derived from 100 replicates. (PNG 912 kb)

High resolution image (TIF 1365 kb)

Table S1

List of primers used in this study. (DOC 37 kb)

Supplementary data 1

All unigenes from the thymus of S. chuatsi. (FA 33487 kb)

Supplementary data 2

Expression analysis and annotation of all unigenes from the thymus of S. chuatsi. (XLSX 3948 kb)

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Chen, X., Shen, Y., Wu, M. et al. Irf3 from mandarin fish thymus initiates interferon transcription. Fish Physiol Biochem 45, 133–144 (2019). https://doi.org/10.1007/s10695-018-0543-8

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