Screening And Functional Study Of Proteins Interaction With The 5’ Untranslated Region Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV)belongs to the Pestivirus genus of the Flaviviridae family,which is the causative agent of bovine viral diarrhea(BVD).BVDV not only infects cattle,sheep,pigs and other livestock,but also infects wild animals such as deer,camels etc.In eukaryotic mRNA,the5’untranslated region(5’UTR)can regulate the translation of mRNA together with RNA binding protein(RBP);The 5’UTR in the BVDV genome plays an important role in the transcription and translation of the virus.However,few studies have been done on BVDV 5’UTR.Which proteins can interact with BVDV 5’UTR and how do they affect BVDV replication remain unclear.In this study,the newly developed RNA-protein interaction detection(Ra PID)technology was used to screen proteins that can interact with BVDV 5’UTR;Knock down the interacting protein by si RNA to explore the effect of the interacting protein on BVDV replication,the study will provide a basis for further elucidating the molecular mechanism of BVDV infection.Amplification of BVDV 5’UTR gene and construction of eukaryotic expression vector1.Amplification of BVDV 5’UTR gene and construction of eukaryotic expression vectorAccording to the BVDV genome sequence in the Gen Bank database,the amplification primers of the BVDV 5’UTR gene were designed,and the BVDV 5’UTR gene was amplified by PCR.The amplified BVDV 5’UTR gene was cloned into an RNA motif vector,and the bacterial liquid was identified by PCR for sequencing analysis.The experimental results showed that: Successfully amplified the BVDV 5’UTR gene and inserted it into the digested RNA motif vector,and successfully constructed the RNA motif-BVDV 5’UTR eukaryotic expression vector.2.Screening proteins interact with BVDV 5’UTR by Ra PID technologyThe constructed RNA motif-BVDV 5’UTR plasmid and BASU plasmid were transfected into HEK-293 T cells to package lentivirus,and the lentivirus was collected to infect MDBK cells and screened for positive cells with puromycin and hygromycin B;RT-PCR was used to detect whether BVDV 5’UTR was stably expressed in the cell line;the above cell lines were treated with different concentrations of Biotin and different times,and Western blot was used to determine the optimal concentration and time of Biotin treatment;The RNA motif-BASU and RNA motif-BVDV5’UTR-BASU cell lines were expanded and treated with the determined optimal concentration of Biotin.The extracted proteins were analyzed by Dynabeads? My One? Streptavidin T1 magnetic bead affinity chromatography.The experimental results showed that: the stable expression cell lines of RNA motif-BASU and RNA motif-BVDV 5’UTR-BASU were successfully constructed;the best effect was when 200 μM Biotin was treated for 18 h;RNA motif-BVDV 5’UTR-BASU was compared and analyzed,and 11 differential proteins were finally screened.3.Functional study of BVDV 5’UTR interacting proteinsThe screened differential proteins were subjected to bioinformatics analysis and related research literatures were reviewed,and two candidate factors,TRAF2 and SNED1,were finally screened for subsequent functional studies.si RNA was designed to knock down expression of TRAF2 and SNED1 genes in MDBK cells,and RT-q PCR was used to detect the expression of TRAF2 and SNED1 genes;After BVDV infection of TRAF2 and SNED1 knockdown cells,RT-q PCR was used to detect the level of BVDV 5’UTR mRNA after BVDV infection at different times;The accumulation of BVDV double-stranded RNA(ds RNA)was detected by immunofluorescence,the cytopathic effect(CPE)of BVDV was observed under an inverted microscope,and the change of virus titer was determined according to Kaber method.The experimental results showed that: RT-q PCR results showed that knockdown of TRAF2 and SNED1 significantly reduced BVDV 5’UTR mRNA levels;After BVDV infection of TRAF2 and SNED1 knockdown cells,the accumulation of green fluorescent-labeled BVDV ds RNA in the cells was reduced,and the CPE phenomenon caused by BVDV infection was weakened,and the progeny virus titer was reduced.In this study,an RNA motif-BVDV 5’ UTR eukaryotic expression vector was successfully constructed,and the proteins interacting with BVDV 5’ UTR were screened by Ra PID technology.si RNA knockdown of TRAF2 and SNED1 genes significantly prevented BVDV replication.This study provides theoretical basis for exploring the mechanism of BVDV infection.