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Study On The Molecular Mechanism Of Bovine Viral Diarrhea Virus Non-structural Protein NS4B Inhibiting Type ? Interferon Production

Posted on:2022-05-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:S YueFull Text:PDF
GTID:1480306320971879Subject:Prevention of Veterinary Medicine
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Bovine viral diarrhea virus(BVDV)was a single-stranded positive-stranded RNA virus.BVDV belongs to the Flaviviridae family,genus Pestivirus member.BVDV was contact infectious disease characterized by diarrhea,fever,leukopenia,oral cavity and digestive tract mucosal erosion and necrosis,miscarriage of pregnant cows or teratogenic fetuses in cattle,acute viral infection can cause immunosuppression,pregnancy 30-120-day-old infection caused persistent infection and immune tolerance in calves.This disease is widespread in cattle farms all over the world,causing serious economic losses to animal husbandry production.The World Organization for Animal Health(OIE)has listed it as a quarantine disease that must be quarantined for import and export animal trade,and my country is classified as a second-class disease.Previous studies have shown that the non-structural protein Npro and the structural protein Erns(E0)encoded by the BVDV genome can inhibit the host's innate immune response.The non-structural protein NS4B plays a key role as a scaffold in viral replication complexes,but little is known about its role in viral pathogenesis.This study mainly explores the role of NS4B protein encoded by the BVDV genome in the process of innate immune response,and clarifies its type I interferon signaling pathway mediated by RIG-I like receptors(RLRs)and its molecular mechanism.In the first place,this study constructed p CDNA3.0-HA-NS4B eukaryotic expression plasmid,amplified the NS4B gene by PCR,and connected the target gene and expression vector after gel recovery and enzyme digestion.After transformation and double enzyme digestion,the target gene and expression vector were connected.The identified correct recombinant plasmid p CDNA3.0-HA-NS4B was transfected into HEK-293T and MDBK cells,and verified by western blot to verify NS4B expression,further use dual luciferase reporter gene,real-time fluorescent quantitative PCR and ELISA methods to verify the effect of NS4B on type I interferon.The results showed that an obvious band of interest appeared at 39 KDa,which proved that the eukaryotic expression vector was successfully constructed.Subsequent use of dual luciferase reporter gene and reverse transcription real-time fluorescent quantitative PCR found that BVDV NS4B significantly inhibited Se V-induced IFN-?promoter activation in a dose-dependent manner,while NS4B inhibited IFN-?m RNA level in HEK-293T cells and MDBK cells.ELISA results showed that NS4B inhibited Se V induced HEK-293T cells supernatant of IFN-?secretion.This study shown that BVDV NS4B has the ability to inhibit type I interferon.Secondly,in order to explore the mechanism by which BVDV NS4B inhibits the type I interferon response mediated by RLRs,through the phosphorylation detection of the downstream signaling molecule IRF3 in the RLRs pathway,it was found that overexpression of BVDV NS4B can inhibit IRF3 phosphorylation in HEK-293T cells.The activation of IFN-?promoter by key signal molecules in the NS4B and RLRs signaling pathways was detected by the dual luciferase reporter gene,and it was found that NS4B significantly inhibited the expression of IFN-?induced by MDA5.Subsequently,overexpressed NS4B,and the transcription level of each signal molecule was detected by real-time fluorescent quantitative PCR.It was found that the m RNA level of MDA5 was reduced in both time periods of 12 h and 24 h after transfection of HEK-293T cells,and 24 h after transfection of MDBK cells also significantly reduced the m RNA level of MDA5.The co-immunoprecipitation experiments revealed NS4B interaction with MDA5,and the laser confocal detection confirmed that NS4B co-localized with MDA5 in the cytoplasm.This study shown that BVDV NS4B inhibits the activation of type I interferon pathway by interacting with MDA5.Finally,in order to further explore which domain of BVDV NS4B interacts with MDA5,the eukaryotic expression vector plasmids with three different domains of MDA5 were constructed.Verification by western blot showed that 2CARD,HEL,and CTD showed obvious target bands at34 KDa,70 KDa and 20 KDa,respectively.Co-immunoprecipitation experiments revealed that BVDV NS4B interacts with the 2CARD domain in MDA5.And laser confocal detection found that NS4B co-localized with MDA5-2CARD in the cytoplasm.In summary,this study proved that the BVDV NS4B protein interacts with 2CARD of the MDA5 domain in the RLRs signaling pathway negatively regulate the expression of type I interferon.It further improves the understanding of the mechanism of BVDV non-structural proteins involved in evading the host's innate immune response,and lays a theoretical foundation for an in-depth understanding of the pathogenic mechanism of BVDV.
Keywords/Search Tags:Bovine viral diarrhea virus, NS4B protein, Innate immunity, RLRs signal pathway, Type ? interferon
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