Abstract
Innate immune system is triggered by pattern recognition receptors (PRRs) recognition. Retinoic acid-inducible gene 1 (RIG-I) is a major sensor that recognizes RNA ligands. However, chickens have no homologue of RIG-I; instead, they rely on melanoma differentiation-associated protein 5 (MDA5) to recognize RNA ligands, which renders chickens susceptible to infection by influenza A viruses (IAVs). Here, we engineered the cMDA5 viral RNA sensing domain (C-terminal domain, CTD) such that it functions similarly to human RIG-I (hRIG-I) by mutating histidine 925 into phenylalanine, a key residue for hRIG-I RNA binding loop function, or by swapping the CTD of cMDA5 with that of hRIG-I or duck RIG-I (dRIG-I). The engineered cMDA5 gene was expressed in cMDA5 knockout DF-1 cells, and interferon-beta (IFN-β) activity and expression of interferon-related genes were measured after transfection of cells with RNA ligands of hRIG-I or human MDA5 (hMDA5). We found that both mutant cMDA5 and engineered cMDA5 triggered significantly stronger interferon-mediated immune responses than wild-type cMDA5. Moreover, engineered cMDA5 reduced the IAV titer by 100-fold compared with that in control cells. Collectively, engineered cMDA5/RIG-I CTD significantly enhanced interferon-mediated immune responses, making them invaluable strategies for production of IAV-resistant chickens.
Key points
• Mutant chicken MDA5 with critical residue of RIG-I (phenylalanine) enhanced immunity.
• Engineered chicken MDA5 with CTD of RIG-I increased IFN-mediated immune responses.
• Engineered chicken MDA5 reduced influenza A virus titers by up to 100-fold.
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All data generated or analyzed during this study are included in this published article (and its supplementary information files).
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Funding
This work was supported by the National Research Foundation of South Korea (NRF) grant funded by the Korea Government (MSIT) (No. 2015R1A3A2033826) and the BK21 FOUR Program of the Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
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SJW, YHP, and JYH designed the research. SJW performed the experiments. HJC and YHP interpreted and reviewed the data. SJW wrote the first draft of the manuscript. HJC, YHP, DR, J-KK, and JYH helped to write the final version of the manuscript. All authors have read and approved the final version of the manuscript.
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Woo, S.J., Choi, H.J., Park, Y.H. et al. Amplification of immunity by engineering chicken MDA5 combined with the C terminal domain (CTD) of RIG-I. Appl Microbiol Biotechnol 106, 1599–1613 (2022). https://doi.org/10.1007/s00253-022-11806-4
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DOI: https://doi.org/10.1007/s00253-022-11806-4