Functional Differentiation Between Grass Carp(Ctenopharyngodon Idella)RIG-I And MDA5 In Interferon Induction

Innate immune response aroused by the recognition of pathogen recognition receptors(PRRs)to pathogen-associated molecular patterns(PAMPs)is the first line of host cells against the invasion of various pathogens.Intracellular virus can be recognized by the intra-cytoplasmic PRRs,namely retinoic acid-inducible gene I(RIG-I)-like receptors(RLRs).Once the recognition has occurred,the downstream signals are activated and interferon regulatory factor(IRF)3 and IRF7 are phosphorylated which leads to the dimerization of IRF3 and IRF7 to induce the production of type I interferon(IFN-I).And ultimately,those antiviral proteins are expressed and adaptive immune system is activated.RIG-I and melanoma differentiation-associated gene 5(MDA5)are the members of RLRs family and play critical roles in the antiviral immune response.Interestingly,RIG-I homologues in the genomes of some fishes(such as large yellow croaker,Pseudosciaena crocea),chicken(Gallus gallus),and Chinese tree shrews(Tupaia belangeri chinensis)cannot be identified,but MDA5 homologues are ubiquitously identified in the studied vertebrates,which may imply that RIG-I and MDA5 are functionally redundant.At present,it is considered that there is difference between RIG-I and MDA5 in the recognition of ligands,and that they are redundant in the recognition to certain virus.However,the difference effect between them on the IFN induction is poorly understood.Grass carp(Ctenopharyngodon idella)is one of the ―Great Four Cultured Fishes‖ in China,whose breeding industry is seriously puzzled by the hemorrhagic disease caused by a double strand RNA(dsRNA)virus,namely grass carp reovirus(GCRV).For this reason,GCRV and the immune system of grass carp have attracted researcher’s attention and concern for a long time.Previously,grass carp RIG-I and MDA5 gene had been cloned and identified to play positive roles in the conflict against GCRV infection.And recently,grass carp IFNs were clearly identified and characterized with the help of high-throughput sequencing,which showed that grass carp IFN-I contained 4 members,namely IFN1,IFN2,IFN3 and IFN4,while grass carp IFN-II contained two members,namely IFN-γrel and IFNγ.It was reported that different fish IFNs play different roles,but little is known about the precision IFN induction pathways and the expression patterns of grass carp IFNs.Thus,we attempt to investigate the relative expression profiles of grass carp IFNs in CIK cells and,more importantly,we will employ GCRV to infect CIK cells to investigate the differential effect between grass carp RIG-I and MDA5 on the induction of IFNs.By real-time quantitative PCR(RT-qPCR),dual-luciferase activity assay,we found that IFN1 and IFN3 were relatively highly expressed,and IFN-γrel and IFNγ were moderately expressed,while IFN2 and IFN4 were relatively lowly expressed in CIK cells without any immunostimulation;that IFNs and representative IFN-stimulated genes(ISGs)were higher expressed in MDA5 overexpressed cells than those in RIG-I overexpressed cells;and that the promoter activities of various IFNs in MDA5 overexpressed cells were higher than those in RIG-I overexpressed cells.In order to examine whether grass carp IRF3 and IRF7 were implicated in this process,we employed cell viability assay,RT-qPCR and immunoblotting(IB)to verify the positive roles of IRF3 and IRF7 in the antiviral immune response of CIK cells.The results indicated that IRF3 and IRF7 were translocated from cytoplasm into the nucleus under GCRV infection or poly(I:C)(a dsRNA analogue)stimulation,though IRF7 was distributed throughout the whole cell;and that IRF3 and IRF7 played positive roles in the anti-GCRV infection immune response.Subsequently,by semi-qPCR,dual-luciferase activity and IB assays,we found that grass carp RIG-I or MDA5 overexpression cannot affect the expression of IRF3 and IRF7 without any immunostimulation,but they positively affected IRF3 and IRF7 at the protein level and phosphorylation level under GCRV infection or poly(I:C)infection.Furthermore,by immunoprecipitation(IP)and IB assays we found that RIG-I or MDA5 overexpression facilitated the serine-phosphorylation levels of IRF3 and IRF7,whilst MDA5 overexpression lead to the up-regulation of threonine-phosphorylation level of IRF7 but RIG-I overexpression impaired it.As a consequence,CiMDA5 enhanced the hetero-dimerization of CiIRF3 and CiIRF7,and homo-dimerization of CiIRF7,whereas CiRIG-I facilitated the hetero-dimerization but attenuated homo-dimerization of CiIRF7.Moreover,to investigate the effects of the dimers of grass carp IRF3 and IRF7 on the promoter activities of various IFNs,the dual-luciferase activity assay was conducted,which suggested that the homo-dimer of IRF3 just enhanced the promoter activity of IFN1,while the hetero-dimer of IRF3 and IRF7 and the homo-dimer of IRF7 were able to induce much more extensive IFN-I responses,emphasizing the critical role of the homo-dimerization of IRF7 and clarifying the molecular mechanism of the difference between RIG-I and MDA5 in IFNs’ induction.At last,we found MDA5 overexpression induced a higher expression of interleukin 4(IL-4)and IL-12p40 genes than RIG-I overexpression,which may be a reason for that MDA5 overexpression induced a stronger IFN-II response than RIG-I overexpression.The present study demonstrates that grass carp MDA5 induces a stronger IFN response than RIG-I in the antiviral immune response,which may be regarded as an evidence to explain the functional redundancy of RIG-I and MDA5.Meanwhile,the present results offer a rational explanation for the observation that RIG-I homologues are lost in some vertebrates without too much effects on the innate immune responses.