Mechanism Exploration Of Non-cytopathic Bovine Viral Diarrhea Virus Inhibiting The Production Of Type ? Interferon

Bovine viral diarrhea-mucosal disease(BVD-MD)is an important bovine infectious disease caused by Bovine viral diarrhea virus(BVDV).Its secrecy,long-term transient infection and persistent infection of animals make BVD-MD rampant in cattle herds all over the world,causing great economic losses to the world cattle industry.NCP BVDV infection can cause severe immunosuppression of the host and secondary infection of other pathogenic microorganisms.The main reason is that NCP BVDV infection can not well induce the innate immune response of cells.Type I interferon(IFN-I)pathway is the main component of innate immunity and plays an important role in the process of organism controlling and eliminating pathogens.However,NCP BVDV infection triggers poor type I IFN responses and the molecular mechanism of its inhibition of interferon expression is still unclear.In order to further clarify the pathogenic mechanism of NCP BVDV and understand the interaction between virus and host,this paper made an exploratory study on the molecular mechanism of NCP BVDV evading IFN-I responses.Objective:(1)Transcriptome sequencing was performed to obtain m RNA librarys of host cells infected with NCP BVDV.Bioinformatics was used to analyze the pathways and cellular processes involved in the enrichment of differentially expressed genes(DEGs)induced by NCP BVDV,in order to lay a foundation for understanding the interaction between virus and host.(2)A Luciferase reporter system of bovine IFN-?(Bo IFN-?)promoter was constructed to analyze the effects of NCP BVDV and its non-structural proteins on the expression interferon and to identify the target of NCP BVDV inhibiting host interferon production.At the same time,it provides a convenient tool for studying the mechanism of virus inhibiting the production of Bo IFN-?in the future.(3)The function of bovine SIKE1(Bo SIKE1)gene upregulated by NCP BVDV was studied,and the relationship between Bo SIKE1 and BVDV proliferation was determined,with the aim to provide evidence for e Lucidation of the mechanism of SIKE1 in innate immune response.Methods:(1)Firstly,bovine peripheral blood mononuclear cells were isolated from bovine peripheral blood cells,and the expression level of monocyte surface antigen CD14 was detected by flow cytometry.Then bovine monocytes were infected with NCP BVDV,and samples were collected at different stages of infection.Construction of RNA-seq library for qualified RNA samples and sequenced in the Illumina Hiseq 2500 sequencing platform.The sequencing results were analyzed by bioinformatics analysis of DEGs,followed by GO and KEGG cluster analysis of DEGs,and q RT-PCR was used to verify whether the transcriptional expression level of the selected IFN-related DEGs was consistent with the RNA sequencing results.(2)The second,the functional element sequence of bovine IFN-?promoter was cloned by molecular biology technology,and the Luciferase reporter system was constructed.The Luciferase activity of NCP BVDV and its non-structural proteins on poly(I:C)-induced IFN-?,NF-?B and IRSE promoter was detected by Luciferase reporter gene analysis.Western blot was used to detect the expression of IRF7,IRF7 phosphorylation and RIG-I protein and in order to verify the effects of NCP BVDV and non-structural proteins on the inhibition of host IFN-?production.(3)Finally,the Bo SIKE1 gene was cloned and expressed,and the corresponding polyclonal antibodies were prepared.The effects of overexpression and interference of Bo SIKE1 on the proliferation of BVDV were verified by q RT-PCR and Western blot.The interaction protein of Bo SIKE1 was studied by LC-MS/MS and Co-IP.Results:(1)After bovine monocytes infected by NCP BVDV,cell immunofluorescence detection showed that NCP BVDV primary cell infection model in vitro was successfully constructed;Transcriptome sequencing analysis showed that more differential expression,either up-or down-regulated was seen at 2 h post-infection(hpi),the expression of 9959 genes was significantly changed compared with those of the controls,of which 4968 genes were upregulated,At 24 hpi,the expression of 7977 genes was significantly different from that of the controls,with 4184 genes up-regulated;Further analysis of immune response pathway related DEGs,compared with DEGs at 2and 24 hpi,the DEGs related to immune response or antiviral activity at 2 hpi was significantly higher than that at 24 hpi;The differential expression profiles of select the type I interferon signaling pathway interferon(IFN)-stimulated genes(ISGs),and genes involved in the innate immune response,including IRF7,DDX3X,TLR13,DDX58(RIG-I),MVAS,TLR9,TRAF6,IRF1,IFIT1,STAT1,ISG20,TRIM25,MX1,NLRX1,CYLD,SIKE1 and ZAP70 were confirmed by real-time quantitative PCR and consistent with the RNA-seq data.(2)The core promoter element sequence of Bo IFN?was cloned and the Luciferase reporter system of Bo IFN?gene promoter was successfully constructed;Bo IFN?3-Luc-MDBK cell line was obtained by screening resistance genes.Bo IFN?3-Luc-MDBK cells were infected with NCP BVDV.It was found that NCP BVDV strain infection could significantly inhibit the transcriptional activity of IFN-?3 induced by poly(I:C)and inhibit the expression of downstream antiviral genes;Seven nonstructural proteins(N^pro,NS2,NS3,NS4A,NS4B,NS5A and NS5B)of NCP BVDV were synthesized and ligated with lentiviral vector.Restriction endonuclease digestion and sequencing showed that the recombinant lentiviral vectors was constructed successfully,and the packaged lentivirus could express the target gene after infection with MDBK;The Luciferase activity of NCP BVDV and seven nonstructural proteins on promoter of Hu IFN-?,Hu NF-?B,Hu IRSE and Bo IFN-?was detected by Luciferase reporting system.The results showed that,NCP BVDV could inhibit the transcriptional expression of IFN-?,and the non-structural proteins N^pro and NS4B could significantly inhibit the transcriptional expression of IFN-?;Overexpression of NS4B protein in HEK293T cells could significantly inhibit the expression and phosphorylation level of IRF7,but had no inhibitory effect on the expression of RIG-I.(3)The ORF of Bo SIKE1 gene was 642 bp.Homology analysis showed that the amino acid similarity of Bo SIKE1 with sheep,pig,human and mouse was 99%,96.1.0%,94.2%and 91.1%,respectively;The prokaryotic recombinant expression plasmid p ET22b-Bo SIKE1 was constructed and successfully expressed in E.coli.Through the optimization of induction conditions,all Bo SIKE1 fusion proteins were expressed in the form of inclusion bodies.The Bo SIKE1 protein was purified and rabbit polyclonal antibodies were prepared successfully.The antibody ELISA titer was more than 1:128000;The effect of Bo SIKE on the proliferation of BVDV was verified by overexpression and interference.Overexpression of Bo SIKE1led to the enhancement of BVDV replication,interference with the expression of Bo SIKE1 would lead to the decrease of BVDV replication,and SIKE inhibited antiviral immunity;LC-MS/MS and Co-IP data showed that there is a direct interaction between Bo SIKE1 and cytoskeleton protein TUBA1D.SIKE may mediate the rearrangement of cytoskeleton needed for innate immunity and act as a bridge between cytoskeleton structure and innate immune signal pathway.Conclusion:(1)The model of bovine primary immune cells infected with NCP BVDV was successfully constructed and the transcriptional differential expression profiles were obtained,and 95DEGs related to IFN-?signal pathway were screened.(2)Through the Bo IFN?promoter Luciferase report system detection screening,NCP BVDV nonstructural protein N^proand NS4B showed specific inhibition on IFN-?(3)Bo SIKE1 can affect the proliferation of NCP BVDV,and there is a direct interaction between Bo SIKE1 and TUBA1D.It is speculated that Bo SIKE1 protein may be a bridge between cytoskeleton structure and innate immune signal pathway.