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IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae

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Abstract

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.

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Acknowledgments

We thank the Zebrafish Core Laboratory of Taipei Medical University for help with the zebrafish experiments.

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Author notes

  1. Chang-Jen Huang and Chih-Ming Chou contributed equally to this work.

Authors and Affiliations

  1. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    Chang-Jen Huang & Huang-Wei Lien

  2. Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, 250, Wuxing St, Taipei 110, Taiwan

    Chih-Ming Chou, Yimin Wu & Chia-Hsiung Cheng

  3. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Chih-Ming Chou, Jhih-Yun Ho & Chia-Hsiung Cheng

  4. Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan

    Cheng-Ying Chu

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  1. Chang-Jen Huang
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Correspondence to Chia-Hsiung Cheng.

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Funding

This work was supported by the Short-Term Visiting Program for Domestic Scholars of Academia Sinica, Taiwan [grant numbers 103-2-2-14-05] and the Ministry of Science and Technology, Taiwan [grant numbers 104-2311-B-038-001].

Conflict of Interest

The authors declare that they have no conflicts of interest.

Electronic Supplementary Material

Figure S1.

Transcription of ISGs gene were induced by poly[I:C] treatment in ZFL cells. The mRNA level of Mxb (A), Mxc (B), Ifnφ1 (C), Ifnφ2 (D), Ifnφ3 (E) induced by 10, 25, 50 μg/ml poly[I:C] and control were measured by qPCR in ZFL cells. *** indicates a significant difference compared with the control (p< 0.001). * indicates a significant difference compared with the control (p< 0.05). (GIF 73 kb)

High Resolution Image (TIFF 192 kb)

Figure S2.

Zebrafish IRF9-S2C induced transcription of ISGs in ZFL cells for 24h. The mRNA level of Mxa (A), Mxb (B), Mxc (C), Ifnφ1 (D), Ifnφ2 (E), Ifnφ3 (F) induced by Zebrafish IRF9-S2C was measured by qPCR at 24h post-transfection in ZFL cells. *** indicates a significant difference compared with the control (p< 0.001). (GIF 70 kb)

High Resolution Image (TIFF 160 kb)

Figure S3.

Transcription of ISGs gene were induced by poly[I:C] treatment in zebrafish larvae. The mRNA level of Mxa (A), Mxb (B), Mxc (C), Ifnφ1 (D), Ifnφ2 (E), Ifnφ3 (F) induced by 10, 25, 50 μg/ml poly[I:C] and control in zebrafish larvae from 24 hpf to 48 hpf were measured by qPCR. * indicates a significant difference compared with the control (p< 0.001). ** indicates a significant difference compared with the control (p< 0.001). *** indicates a significant difference compared with the control (p< 0.001). (GIF 84 kb)

High Resolution Image (TIFF 203 kb)

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Huang, CJ., Chou, CM., Lien, HW. et al. IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae. Mar Biotechnol 19, 310–319 (2017). https://doi.org/10.1007/s10126-017-9752-x

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