Amplification of immunity by engineering chicken MDA5 combined with the C terminal domain (CTD) of RIG-I

• Published in: Applied microbiology and biotechnology Vol. 106; no. 4; pp. 1599 - 1613
• Main Authors: Woo, Seung Je, Choi, Hee Jung, Park, Young Hyun, Rengaraj, Deivendran, Kim, Jin-Kyoo, Han, Jae Yong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-02-2022; Springer; Springer Nature B.V
• Subjects: Animals; Applied Microbial and Cell Physiology; Aquatic birds; Biomedical and Life Sciences; Biotechnology; Chickens; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Domains; Ducks; Gene expression; Genetic aspects; Histidine; Homology; Humans; Immune response; Immune system; Immunity; Immunity, Innate; Influenza; Influenza A; Innate immunity; Interferon; Interferon-beta - genetics; Interferon-Induced Helicase, IFIH1 - genetics; Life Sciences; Ligands; Melanoma; Methods; Microbial Genetics and Genomics; Microbiology; Mutants; Pattern recognition; Pattern recognition receptors; Phenylalanine; Poultry; Residues; Retinoic acid; Ribonucleic acid; RNA; RNA sequencing; Transfection; β-Interferon; South Korea; RIG-I; Influenza A virus; RNA ligands; MDA5; Chicken
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-022-11806-4

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
h
RIG-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
h
RIG-I or
human
MDA5 (
h
MDA5). 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.