Mechanism Of Cell Cycle Arrest Mediated By Mink Enteritis Virus NS1 Protein

Mink viral enteritis（MVE）is a severe and acute infectious disease of minks caused by mink enteritis virus（MEV）,with a high morbidity and mortality.MEV has spread rapidly since it was first isolated and identified in mink farms in China in 1984,and has been harming the healthy development of mink breeding industry in China,causing significant economic losses.MEV is a self-replicating virus that cannot synthesize DNA polymerase and topoisomerase by itself.MEV requires the host cell to provide the corresponding cell replication factor.MEV NS1 protein is a multifunctional protein with ATPase and helicase activities.MEV NS1 protein can specifically bind to DNA replication origin and play a very important role in the replication of viral DNA.The purpose of this study is to analyze the mechanism of cell cycle arrest induced by MEV NS1 protein,exploring the role of nuclear localization signal and trans-activated domains in NS1 protein induced cell cycle arrest.This experiment takes MEV-SD7 strain isolated and preserved in our laboratory as the research object.Using eukaryotic expression vector p CMV-Myc and MEV-SD7,we constructed p CMV-Myc-NS1（p NS1）to express MEV NS1 protein,p CMV-Myc-NS1m NLS△586-654（p NS1m NLS）to express NS1 protein without nuclear location signal（NLS）and p CMV-Myc-NS1m TAD△1630-2007（p NS1m TAD）to express NS1 protein without trans-activated domain（TAD）.We set up MEV infection group,p NS1 transfection group,p NS1m NLS transfection group,p NS1m TAD transfection group,p CMV-Myc empty vector transfection group and blank control group.The digested CRFK cells were seeded in the 6-well cell culture plates.After 12 hours of culture,CRFK cells were synchronized in G₀ phase by serum starvation method.Synchronous infection of 1.0 MOI MEV in cells of MEV infection group,transfection of 5μg/well recombinant plasmid described above in cells of each transfection group.Using the polyclonal antibody prepared in this experiment to detect the expression of NS1 protein in each group.The results showed that NS1 protein was expressed in MEV infection group,p NS1 transfection group,p NS1m NLS transfection group and p NS1m TAD transfection group.Flow cytometry was used to detect whether MEV NS1 protein caused host cell cycle arrest.The results demonstrated that MEV increased the proportion of CRFK cells in S phase and induced S phase arrest in CRFK cells,and complete NS1 protein also led to cycle arrest in S phase of CRFK cells.And the deletion of nuclear localization signal and trans-activated domain significantly weakened the ability of NS1 protein to cause the S cell cycle arrest.Therefore,NS1 protein plays an important role in the process of S-phase arrest caused by MEV,and nuclear localization signal and trans-activated domain are necessary to the process of cell cycle arrest caused by NS1 protein.Western blotting method was used to detect the expression of CDK2,Cyclin E1,Cyclin A2,p53,and CDC25A in CRFK cells in each group,exploring the mechanism of cell cycle arrest induced by MEV NS1 protein.The results showed that the expression of cyclin E1 was up-regulated in MEV infection group and p NS1 transfection group,and there was no significant difference between other groups and the control group.The up-regulation of cyclin E1 would accelerate the process of cells entering S phase,contributing to MEV NS1 protein to induce S phase arrest.MEV infection and p NS1 transfection also down-regulated the expression of Cyclin A2,which would prevent cells from completing the S phase and entering the next phase.The expression of p53 also increased in these two groups,but the expression of CDC25A did not significantly change.Interestingly,the expression of CDK2 in MEV infection group and p NS1 transfection group was down-regulated.CDK2 can not only form a complex with Cyclin E1 to take charge of the entry of S phase,but also form a complex with Cyclin A2 to take charge of the completion of S phase,so the regulation mechanism of CDK2is relatively complex.The phosphorylation of CDK2 at Thr160 was also analyzed.The results showed that the phosphorylation level of CDK2（p Thr160）in MEV infection group and p NS1transfection group was up-regulated,which was contrary to the down-regulation of CDK2,indicating that although MEV NS1 protein downregulates the expression of CDK2,it promotes the phosphorylation of CDK2 at Thr160.However,there was no significant change in the expression of CDK2,Cyclin E1,Cyclin A2,CDC25A and the phosphorylation of CDK2 at Thr160 in p NS1m NLS transfection group and p NS1m TAD transfection group.This is consistent with the flow cytometry results of the p NS1m NLS transfection group a nd the p NS1m TAD transfection group.In summary,MEV can cause S-phase arrest of CRFK cells and NS1 protein alone also has the ability to cause cell cycle arrest.Nuclear localization signal and trans-activated domain are necessary to the process of cell cycle arrest caused by NS1 protein.MEV and its NS1 protein can up-regulate the expression of Cyclin E1 and p53,down-regulate the expression of Cyclin A2 and CDK2,and promote the phosphorylation of CDK2 at Thr160.However,NS1 protein without nuclear localization signal and trans-activated domain could not cause significant changes in the expression of CDK2,Cyclin E1,Cyclin A2,CDC25A and the phosphorylation of CDK2 at Thr160.This study enriched the research data of MEV molecular biology and provided the theoretical basis for more effective prevention of mink viral enteritis.