Preliminary Study On The Molecular Mechanism Of NF-?B Signaling Pathway Activated By Bovine Viral Diarrhea Virus NS5B Protein

Bovine viral diarrhea(BVD)is an infectious disease mainly caused by the bovine viral diarrhea virus(BVDV),which can cause diarrhea,miscarriage,reproductive disorders and immunosuppression in sick animals.Because of its great harmfulness,the World Organization for Animal Health(OIE)has classified it as a Class B infectious disease,and China has classified it as a Class II infectious disease,but the specific pathogenic mechanism is not fully clear.Therefore,understanding its pathogenic mechanism is essential to curb the spread of the disease.The previous research of our group had confirmed that BVDV NS5 B could significantly activate the NF-?B signaling pathway.To further explore its activation mechanism and clarify the functional domain of NS5 B activating the NF-?B signaling pathway,NS5B protein was truncated into 6 segments for expression: NS5B-core(core area),NS5B-finger(finger area),NS5B-palm(palm area),NS5B-thumb(thumb area),NS5B-N(N terminal),NS5B-c domain(C terminal).Meanwhile,each truncated fragment pCAGGS was constructed the eukaryotic expression vector and transfected into HEK-293 cells.The dual luciferase reporter system preliminarily determined that the functional domains of NS5 B activating the NF-?B signaling pathway were located in the "finger" and "palm" structure areas.Then,we constructed the eukaryotic expression of each truncated fragment pEGFP,and further proved that the "finger" area and "palm" area of NS5B could activate NF-?B,while other structure domains could not activate this signaling pathway by using laser confocal detection and Western blot.Based on this research,we transfected pEGFPNS5 B,pEGFP-NS5B-finger,and pEGFP-NS5B-palm into HEK-293 cells respectively,and extracted 6h,12 h,24h,36 h,48h cell proteins.The results of Western blot showed that compared with the control group,after the activation of the NF-?B signaling pathway,the expression levels of TLRs and RLRs receptors and related linker molecules showed different trends over time,but the expression levels all increased.We constructed the pGBKT7-NS5 B bait plasmid,screened out the host protein that interacts with NS5 B in the bovine kidney cell cDNA library by yeast two-hybrid technology,and verified it by four methods: point-to-point experiment,backcross experiment,laser confocal,and immunoprecipitation.All of these experimental methods were used to clarify the interaction between ARHGEF18 and NS5B.The recombinant plasmid pEGFP-ARHGEF18 was constructed and co-transfected with pEGFP-NS5B into HEK-293 cells.With the two separate transfection as the comparison,the phosphorylation level of p65 was detected by western blot to indicate that ARHGEF18 promotes the activation of NS5 B on NF-?B.