N6-Methyladenosine Modification Regulatesin NS5A Expression Of Bovine Viral Diarrhea Virus

Bovine Viral Diarrhea Virus(BVDV)belongs to flaviviridae,plague virus and single strand RNA virus.NS5A is a kind of virus nonstructural protein with hydrophilic activity.NS5A in flaviviridae members plays a certain role in virus life cycle through phosphorylation of serine and threonine residues.It is reported that m6Amethylation exists in many loci of flaviviridae virus genome,such as yellow fever virus and hepatitis C virus.m~6A loci are enriched in NS3 and NS5 regions,and potentially conserved m~6A loci can regulate the virus maturation process.The research shows that m~6A can mechanically regulate the production of Hepatitis C virus(HCV)particles,and knocking down m~6A methyltransferase like 3(METTL3)and methyltransferase like 14(METTL14)significantly increases the abundance of HCV NS5A protein.On the contrary,the expression of NS5A protein decreased significantly after knocking down m6Ademethylase fat mass and obesity-associated protein(FTO).Because BVDV NS5A and HCV NS5A are very similar in structure and function,it is speculated that m6A can also regulate the expression level of BVDV NS5A protein,and then affect the production of virus particles.In this study,the influence of BVDV replication on m6A methylation modification and the relationship between BVDV NS5A protein and m~6A methylation protein were discussed,which laid a foundation for further understanding the molecular mechanism of m~6A methylation modification regulating virus replication.At first,in order to determine the changes of m~6A methylation level in MDBK cells infected with BVDV,the transcription and expression levels of methyltransferases METTL3,METTL14and demethylase FTO were detected by q-PCR and Western-blot.And the transcription levels of methylation binding protein YTH domain protein YTHDF1(YTH domain-containing family protein 1),YTHDF2(YTH domain-containing family protein 2)and YTHDC1(YTH domain-containing protein 1).The results showed that the transcription and protein expression levels of METTL3 decreased,FTO increased,YTHDF1 decreased,YTHDF2 and YTHDC1increased after CP and NCP BVDV infection.Studies have shown that CP and NCP BVDV infection affects the expression of m~6A methylated protein.Secondly,the eukaryotic expression plasmids pc DNA3.0-METTL3,pc DNA3.0-METTL14and pc DNA3.0-FTO were constructed by PCR amplification,gel recovery,double digestion to identify the target gene and expression vector,ligation and transformation.The recombinant plasmid correctly identified above was transfected into 293T cells by calcium phosphate method and verified by Western blot.The results showed that there were obvious target bands at 64KDa,52KDa and 58KDa,which were consistent with the expected size.The research indicated that the eukaryotic expression vectors of bovine METTL3,METTL14 and FTO protein were successfully constructed,and the bioinformatics analysis of bovine METTL3 and FTO protein laid a foundation for subsequent research.Thirdly,the interaction between bovine METTL3 protein and NS5A protein was predicted by bioinformatics software,and the interaction relationship was verified by Co-IP and other methods.The predicted results showed that the interaction between METTL3 protein and NS5A protein was possible.Co-transfection of NS5A and METTL3 plasmid into 293T cells was carried out by Co-IP method.The results showed that METTL3 protein and BVDV NS5A protein were coprecipitated.The interaction between BVDV NS5A protein and methyltransferase METTL3was preliminarily proved.In 293T cells,overexpression of methyltransferase METTL3 protein can reduce the expression of BVDV NS5A protein,and overexpression of BVDV NS5A protein can reduce the expression of methyltransferase METTL3 protein and demethylase FTO protein.In MDBK cells,overexpression methyltransferase METTL3,suppresses viral replication.In summary,we have demonstrated that CP and NCP-type BVDV infection in MDBK cells can affect the expression level of m~6A methylated protein.BVDV NS5A protein interacted with methyltransferase METTL3.Over-expression of METTL3 in 293T cells decreased the expression of NS5A protein,and over-expression of FTO increased the expression of NS5A protein.Overexpression of BVDV NS5A protein resulted in a decrease in the expression of METTL3 and an increase in the expression of FTO.In MDBK cells,overexpression methyltransferase METTL3,suppresses viral replication.This study laid the foundation for indepth understanding of the function of BVDV NS5A protein and the molecular mechanism of m~6A methylation modification in regulating virus replication.