Antiviral Signaling Pathway And Expression Regulation Of Bovine Novel Type Ⅰ Interferon Subtypes(IFN-ε,IFN-κ,IFN-χ)

Interferon（IFN） is a large class of multi-effect cytokines in all jawed vertebrates,which are mainly responsible for the host’s antiviral immune response.Different pathogen can induce the expression of specific IFN members,which make finding and identifying new members of interferon significant for antiviral immune response.Innate immunity is the first line of defense against the pathogen invasion.Antiviral immune responses is initial with intracellular pattern recognition receptors（PRRs）to identify highly conserved pathogen-associated molecular patterns（PAMPs）expressed on invasive pathogens,then PAMPs recognized by PRRs induce the production of type I IFN by transcription factor NF-κB and interferon regulatory factors（IRFs）（especially IRF3 and IRF7）,establishing the antiviral state to induce adaptive immune responses,and ultimately remove the viral infection from body,which make type I IFN playing an important role for host cells in establishing antiviral infection and resisting viral replication.In this study,bovine type I interferon gene locus were systematically analyzed,and two new subtypes of interferon（IFN-ε,IFN-κ）and a new subtype with two members（IFN-χ1,IFN-χ3）were mapped,then a series of research focusing on the antiviral signaling pathway and expression regulation of these four interferon were conducted.The specific research contents are as follows:1.Construction and analysis of bovine type I interferon gene locus map.A variety of BLAST methods（BLAST,Basic Local Alignment Search Tool）online were used to screen the whole genome of bovine,and the information of all types of interferon was analyzed comprehensively.It was found that the type I interferon gene were all arranged on chromosome8 and have evolved the new family with distinct characteristics,and the IFN-χ subfamily undoubtedly becomes the most noticeable,which has all the characteristics to be a new interferon and is composed of four members.The loci were divided into two large sub-loci according to the transcriptional direction,the length（746 kp）of bovine type I interferon gene locus was twice as long as that of human（403 kp）and murine（323 kb）,and the number of IFN genes（64）was also far more than that of human（17）and murine（19）.At the end of the locus,five IFN-δ pseudogenes and four IFN-χ-like molecules were mixed among 12 IFN-β-like genes,and the transcriptional directions of these genes were the same.The other regions of the locuswere supplemented with a large number of IFN-α（13）and IFN-ω（24）family.IFN-κ and IFN-εonly have one copy sequence located at the beginning of the genome,and the transcriptional direction of IFN-κ is opposite to that of IFN-β,IFN-κ gene is 6.561 Mb apart from the nearest IFN gene.2.Characterization and antiviral signaling pathway of bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3.In this study,the bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 gene were cloned from bovine genome extracted from liver,and the characteristics of these interferon sequences were analyzed.Then the peptide sequences of bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 were expressed in Escherichia coli,and polyclonal antibody against bovine IFN-ε,IFN-κ,IFN-χ was prepared,the antiviral activity of bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 was determined on different cells.The antiviral activity of recombinant bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 were sensitive to trypsin and had some resistance to acid（p H 2.0 and p H10.0）and heat（42 ℃ and 60 ℃）.This antiviral activity can be neutralized by specific polyclonal antibodies and can also be blocked by type I interferon receptor IFNAR1 and IFNAR2 antibodies.Recombinant bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 can induce the transcription of ISGs（Mx1,ISG15,ISG56）,and the expression of Mx1,STAT1 and NF-κB p65.The above results show that known type I interferon members signal through JAK-STAT pathway to exert antiviral activity.3.Effect of bovine IFN-ε,IFN-κ,IFN-χ1,IFN-χ3 on interferon signaling pathway.In this study,transcriptome sequencing was used to analyze the regulated genes of bovine IFN-αA and IFN-χ1 on the primary bovine testis cells,and then the express difference of these genes were compared,the result showed Bo IFN-χ1 treated group has 40 differentially expressed genes up-regulated,and Bo IFN-αA treated group has 80 genes up-regulated and one gene down-regulated.The expression of OAS,STAT1,LGP2,GBP4,BST2,PML gene were selected to verify these selected genes all have different degrees of upregulation.In addition,bovine type I interferon not only can activate the luciferase activity expressed by the commercialized NF-κB-Luc and ISRE-Luc luciferase reporter vectors on BL and BT cells,but also can activate the promoter activity of bovine IFN-β and IFN-χ constructed in this study.The above results give the foundation for the further study of bovine innate immune signaling pathway.4.Study on the related molecules in bovine interferon signaling pathway.RIG-I and MDA5 is an important pattern recognition receptor for intracellular recognition of dsRNA.IRF3 and IRF7 are important transcription factors involved in the innate immune response.The transcription factor IRF9 is involved in the production of type I interferon.ELF4,MAVS and MITA are used as signal transduction adaptor molecules involving in the production of type I interferon.In this study,the c DNA of bovine adaptor molecules ELF4,MAVS,MITA were cloned from the bovine primary kidney cells by RT-PCR,and then the sequence characteristic,tissue expression,cellular location and the effect on interferon signaling pathway were analyzed.ELF4,MAVS and MITA not only can induce the production of different type I interferon,the transcription and expression of related molecular in signaling pathway on different cells,butalso can effectively promote ISRE binding elements,NF-κB elements,IFN-β promoter and IFN-χ promoter activity.The above results lay a basis on the further research on the role of adaptor molecules ELF4,MAVS,MITA in bovine infectious diseases.In addition to the ELF4,MAVS,MITA gene,the important molecules MDA5,IRF3,IRF7,IRF9 and DAI in bovine interferon signaling pathway were cloned,which not only lay the foundation for the study of bovine innate immune,but also supply the tools for the research on the interaction between pathogens and host.5.Effect of 3C^pro,Lbpro on interferon signaling pathway in bovine cells.In this study,the effects on the production of type I interferon and transcription of related molecular in interferon signaling pathway stimulated by different virus or reagents were analyzed,combining with the studies reported previously that FMDV L^pro and 3C^pro can antagonize the host type I interferon response,FMDV target different molecules to escape the host’s innate immune and antagonize the production of type I interferon.In this study,further studies showed that both 3C^pro and L^pro could not only inhibit the production of type I IFN and the transcription of ELF4 and MAVS,RIG-I-like receptor RIG-I and MDA5,transcription factor IRF3 and IRF7,but also suppress the facilitation of type I interferon and adaptor molecule ELF4,MAVS activation of NF-κB,ISRE,Bo IFN-β 、 Bo IFN-χ promoter activity.Western Blot results showed that 3C^pro and L^pro could inhibit the expression of NF-κB p65,Mx1,IRF3,IRF7,ELF4 and MAVS induced by poly（I: C）and Bo IFN-β on BT and MDBK cells.In conclusion,this study analyzed the molecular characteristics and biological activity of new types of type I interferon（IFN-ε,IFN-κ and IFN-χ）,and established a technical platform for the research on bovine interferon signaling pathway,the effect of ELF4,MAVS,MITA on the interferon signaling pathway was systematically analyzed using the established platform in this study,then FMDV 3C^pro,Lbpro was analyzed to evade the innate immune response,which provided the theoretical basis and material support for the development of virus immunization and antiviral reagent.