The Functional Of Cyprinidae Fish IL-27 And IFNd In Host Resistance To Viral Infection

When the host is invaded by pathogens,immune cells produce a large number of cytokines to maintain homeostasis.Spring viremia of carp virus(SVCV)is an important pathogen that infects Cyprinid fishes.Spring viremia virus disease has the characteristics of fast onset,high infection rate and high mortality,which greatly hinders the sustainable development of aquaculture in our country.Interleukin(IL)27,an important member of the IL-12 family,is composed of IL-27A and the Epstein-Barr virus(EBV)inducible gene 3(EBI3)subunit and plays an important role in host inflammation regulation and antiviral immune response.Among them,IL-27A and EBI3 subunits not only function in the form of dimer IL-27,but also have their own unique biological activities and participate in host immune regulation.In this study,two differentially expressed genes,IL-27A and EBI3,were screened and obtained by analyzing the transcriptome data of zebrafish caudal fin cells(ZFIN)infected by SVCV,and their functions were explored as research objects.IL-27A and EBI3 were identified by bioinformatics analysis including amino acid sequence alignment.Double stranded RNA(dsRNA)virus mimics poly(I:C),SVCV,SVCV structural protein and zebrafish(Danio rerio)interferon recombinant protein(IFNφ1)was used to stimulate zebrafish cells.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of IL27A and EBI3 in zebrafish.The effects of IL-27A and EBI3 on SVCV replication were analyzed by qRT-PCR and crystal violet staining,and the role of IL-27A and EBI3 in antiviral immune response of zebrafish was further elaborated.Type Ⅰ Interferon(IFNs)is a key cytokine for host to resist virus invasion.As an important member of the type I IFNs family,IFNd is widely found in teleost fishes.However,the potential receptor of IFNd and its mediated signaling pathway as well as its antiviral activity in cyprinid fishes have not yet been identified,and its crystal structure has not yet been resolved.Based on this,this study took grass carp(Ctenopharyngodon idella,Ci)IFNd(CiIFNd)as the research object.Through obtaining CiIFNd eukaryotic protein and stimulating grass carp primary head kidney cells and epithelioma papulosum cyprinid(EPC)cells,qRT-PCR was used to detect the effect of CiIFNd on host antiviral related genes.The effect of CiIFNd on SVCV replication was detected by crystal violet staining and SVCV infection.The potential receptors and mediated signaling pathways of CiIFNd were analyzed by immunoprecipitation(Co-IP)and phosphorylation assay.The crystal structure of CiIFNd was analyzed and the key functional sites were predicted by crystal diffraction and molecular replacement.Thus,the role of CiIFNd in the process of resisting virus invasion of grass carp was further elaborated,providing theoretical support for the prevention and treatment of fish viral diseases.The results obtained in this study are as follows:1.The functional study of zebrafish IL-27 in host defense against viral infection(1)Bioinformatics analysis of IL-27A and EBI3 in zebrafishZebrafish IL-27A is a cytokine encoded by 292 amino acids,containing signal peptides and 4α long helical structures,and has the conserved amino acid residues Trp89 and Trp261 of other species of IL-27A,which correspond to human IL-27A residues of Trp97 and Trp197,respectively.Bioinformatics analysis showed that the low homology of IL-27A in zebrafish with other species,suggesting that IL-27A might not be conserved during evolution.Zebrafish EBI3 is a cytokine encoded with 302 amino acids.In addition to the signal peptide,EBI3 also has immunoglobulin(IG)and fibronectin type Ⅲ(FN Ⅲ)domains.Bioinformatics analysis showed that zebrafish EBI3 was closely related to cyprinidae fish,but far from salmonidae fish and mammals.It indicates that EBI3 may not have been conserved in the evolution process from fish to mammals.(2)Expression pattern and antiviral activity analysis of IL-27A and EBI3 in ZebrafishWe used SVCV infection or poly(I:C)transfection to detect the expressions of IL27A and EBI3 in ZF4 cells at different time points,and the results showed that the expressions of both were significantly up-regulated.In addition,the expression of IL27A and EBI3 could also be significantly induced after transfecting five structural proteins of SVCV(SVCV-N,SVCV-P,SVCV-M,SVCV-G and SVCV-L),indicating that the expression of IL-27A and EBI3 in zebrafish was induced by SVCV.However,IL-27A and EBI3 are not regulated by IFNφ1.Overexpression and crystal violet staining showed that both IL-27A and EBI3 could significantly inhibit the expression of SVCV-N and SVCV-G genes,but overexpression of IL-27A had no significant effect on the expression of IRF3,IFN,ISG15 and MX1.These results suggest that IL-27A participates in host antiviral immune response in an IFN independent manner.In addition,the co-expression experiment of IL-27A and EBI3 showed that the coexpression of IL-27A and EBI3 had more significant inhibitory effect on SVCV-N and SVCV-G genes than that of IL-27A and EBI3 alone.In conclusion,both IL-27A and EBI3 of zebrafish have antiviral activity,but both are weaker than IL-27.2 Study on the function,structure,and mechanism of action of CiIFNd(1)Expression regulation and antiviral activity analysis of CiIFNdWe obtained CiIFNd eukaryotic protein in human embryonic kidney 293 suspension cells(HEK293F).The protein activity analysis experiment showed that CiIFNd could induce the expression of antiviral genes in grass carp primary head kidney cells and EPC cell lines,but could not induce the expression of inflammatory related factors in the primary head kidney cells of grass carp,indicating that C/IFNd eukaryotic protein had activity and could induce the host antiviral response.Antiviral function experiments showed that CiIFNd could significantly inhibit the transcription of SVCV-N and SVCV-G genes and improve the expression of host antiviral gene MX1.However,compared with CiIFNa,its ability to inhibit SVCV replication was lower.In conclusion,CiIFNd has antiviral activity and can inhibit SVCV replication,but its activity is weaker than CiIFNa.(2)Analysis of potential receptors and mediated signaling pathways of CiIFNdIn teleost fish,type I IFNs receptors are composed of cytokine receptor family B1(CRFB1),CRFB2 and CRFB5.The immunocoprecipitate(Co-IP)experiments showed that CiIFNd could bind with CRFB1,CRFB2 and CRFB5.In addition,there are two STAT1 homologies(STATla and STAT1b)exist in teleost fish due to the whole-genome replication(WGD)event.Phosphorylation analysis results showed that CiIFNd could activate the phosphorylation of STAT1a,STATlb and STAT2.Among them,the phosphorylation level of STATlb is low.In conclusion,CiIFNd activates JAK-STAT signaling pathway for signal transduction by binding with receptors CRFB1,CRFB2 and CRFB5.(3)Analysis of crystal structure and key functional sites of CiIFNdBy X-ray diffraction,we determined that the resolution of the CiIFNd crystal structure is 1.91 ? and the space group is P1211.The CiIFNd contains six alpha helices(A-F),whose structural core consists of A,C,D and F helices folded with each other,and has the typical characteristics of type I IFNs(F helix is straight helix).In the structure of CiIFNd,there is a disulfide bond composed of Cys20 and Cys115 residues,which connects the N-terminal with the D-helix.In addition,compared with other typeⅠ IFNs,CiIFNd and CiIFNa have the most similar structure,with a standard score(Zscore)of 19.6.The structural modeling and antiviral function analysis showed that the R94,I131 and K134 amino acid residues of CiIFNd may be the key sites for its antiviral function.