Study On Regulation Of Bovine Viral Diarrhea Virus Proteins In Its Non-coding Region

Bovine Viral Diarrhea,also called mucosal disease,is caused by Bovine Viral Diarrhea Virus(BVDV).It will also bring huge economic losses to the cattle industry around the world and has been classified as a B class infectious disease by the World Organisation for Animal Health(OIE).Bovine viral diarrhea virus(BVDV)is a member of the Pestivirus genus of the Flaviviridae family,its diamete is 25 to 30 nm.It is an single positive-stranded RNA virus with enveloped memberane,genome size is about 12.3 kb.It is composed of 5’non-coding region,open reading frame(ORF)and 3’ non-coding region.The ORF can encode a protein polymer containing nearly 3900 amino acids,and then it can be cut into 12 protein,which were successively named Npro,C,Erns,E1,E2,P7,NS2,NS3,NS4 A,NS4B,NS5 A and NS5 B.Of these proteins,C,Erns,E1 and E2 are structural proteins,forming viral particles together with genomic RNA and lipid membranes.And they are important for the interaction between the virus and the receptor.The remaining eight are non-structural proteins that play a role in the process of virus replication.The non-coding region of bovine viral diarrhea virus(BVDV)plays a major role in the initiation and stability of RNA translation: 5 ’-the Internal ribosome entry site(IRES)can guide the initiation of polyprotein synthesis.3 v in the 3 ’end non-coding motif is a collaborative activity sequence and structure components of complex RNA motif that plays an important role in the viral RNA replication and termination of translation.Firstly,this study identifies the genotype of a BVDV virus isolate and has found it is type I BVDV.This virus and Singer_Arg(GenBank: DQ088995.2)share a sequence consistency of 99%.And then,according to available genomic sequences of Singer_Arg,and BVDV I complete genome,we acquired the whole genome sequenceof the BVDV Singer_Arg vatiant virus.Secondly,according to the whole genome sequence of BVDV virus,we designed12 proteins and two non-coding primers,Then,with the help of PCR,gene sequencing technology and other technologies,we successfully obtained the interest fragment.And these 12 proteins were cloned into the eukaryotic expression vector pCMV-HA to become protein eukaryotic expression plasmid.At the same time,two non-coding genes were connected to luciferase expression vector pMIR-report to become luciferase expression plasmid.Finally,at the level of translation,we first tested the activity of bovine viral diarrhea virus 3 ’UTR in PK15 cells,the results showed that 3 ’UTR of bovine viral diarrhea could significantly inhibit the translation efficiency.Then we explored the regulating effect of proteins of bovine viral diarrhea virus on its 3 ’UTR.Through the cell co-transfection technique,we first introduced the eukaryotic expression plasmids of the 8 proteins of the bovine viral diarrhea virus and the luciferase expression plasmid of its 3’UTR into PK15 cells,respectively.Then,we examined which proteins regulate the function of 3 ’UTR according to the determination of luciferase.The results showed that bovine viral diarrhea virus NS2,NS4 B,NS5A,C and Erns protein had obvious regulatory effects on its 3 ’UTR.Among them,NS4 A,NS4B,C and Erns protein can significantly reduce the translation inhibition function of 3 ’UTR.Therefore,we suggest that NS4 B,NS5A,C and Erns protein can promote the translation of virus.However,NS2 protein significantly enhanced the translation inhibition function of 3 ’UTR,So we suggest that NS2 protein can blocks the translation of the virus through reaction with 3 ’UTR.In conclusion,in this study,we found that bovine viral diarrhea virus NS2,NS4 B,NS5A,C and Erns protein could significantly adjust the translation of 3 ’UTR by using the dual-luciferase reporter gene detection system.