Immunologicfunction Of MDA5 And The Functional Mechanism Of IRF3/7 In MDA5 Signaling Pathwayin Grass Carp(Ctenopharyngodon Idella)

Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)results in tremendous loss of grasscarp cultivation,immune prophylaxis and treatmentmay be the most effective solution for this disease. However, this solution is strongly restricted by the immumological research on fishes. Melanoma differentiation associated gene 5(MDA5) belongs to retinoic acid-inducible gene-I like receptors(RLRs) family,after viral infection, MDA5 recruits IFN-β promoter stimulator 1(IPS-1) by CARDs domainand thenactivates TANK-binding kinase 1(TBK1).Subsequently,activated TBK1 leads to the phosphorylation of interferon regulatory factor 3(IRF3)and IRF7 and ultimately induces the expression of type I interferons(IFN-I). IRF3 and IRF7 are transcriptional factors in RLRs signaling pathway and play an important role in the regulationof IFN-I.In this study, using DNA recombinant technique, the expression vectors forctenopharyngodon idellaMDA5(CiMDA5) orits domainswere constructed and the immunologic function of CiMDA5 and the functional mechanism of Ci IRF3 and CiIRF7 in CiMDA5 signaling pathway were clarified at the cellular level after transfected these vectors to ctenopharyngodon idellakidney(CIK) cells.The main results were listed as follows:1. CiMDA5 or CiMDA5 domainsstably expressedCIK cells were obtained.2. Overexpression of CiMDA5 or CiMDA5 domainscould protect CIK cells from GCRV infection and repress the replication of GCRV in CIK cells.3. CiMDA5 could respond to GCRV, LPS or PGN stimulationand enchance the expression of CiIPS-1, CiIFN1, CiIL-1βandCiMx1.4. AfterGCRV, LPS or PGN stimulation, CARD could induce signaling cascade, Helicase or RD negatively regulatedthe function of CARD by intramolecular interaction, RD enhanced the function of CiMDA5 by intermolecular interaction.5. After poly(I:C) stimulation, overexpression of CiMDA5 repressed the expression of CiIPS-1, CiIFN1,CiIL-1β andCiMx1, overexpression of CARD, RD, ΔCARD or ΔRD slightly enchanced the expression of CiIFN1 and CiMx1.6. CiIRF3 and CiIRF7 participate in CiMDA5 signaling pathway by protein phosphorylation.Taken together, this study revealed the immunologic function ofCiMDA5 and CiMDA5 domainspost GCRV, poly(I:C), LPS or PGN stimulation.In addition,it is also found that CiIRF3 and CiIRF7 simultaneously participate in CiMDA5-mediated antiviral innate immunity pathwaysby protein phosphorylation. These results wouldlay a foundation on the futher research of fish innate immunity and exploration oftheimmune prophylaxis and treatment of the grass carp hemorrhagic disease.