Molecular Mechanism Underlying Non-structural Protein NS1 Of Mink Enteritis Virus Induced Apoptosis

Mink viral enteritis(MVE)is a highly contagious and lethal disease caused by mink enteritis virus(MEV),which is characterized by high morbidity and mortality,particularly the newborn mink.The MEV genome contains two main open reading frames,which encode non-structural proteins NS1 and NS2 and structural proteins VP1 and VP2,respectively.Non-structural protein NS1 regulates the replication of MEV and transactivated viral structural proteins expression.Since it has been reported that NS1 of parvovirus has a cytotoxic effect and can induce apoptosis.However,the molecular mechanism of apoptosis modulated by MEV and its NS1 is still unclear.The purpose of this study is to analyze the signaling pathway and mechanism of apoptosis induced by MEV and its NS1 protein,and to lay a theoretical foundation for revealing the pathogenic mechanism of MEV infected mink.In order to reveal whether MEV can induce apoptosis in vivo and in vitro,we infected healthy minks with MEVB strain and analyzed the apoptosis of various tissues of the digestive tract.The results indicated that the infected mink had different degrees of pathological changes in the digestive tract tissues.Apoptotic cells were found in the esophagus,small intestine,mesenteric lymph nodes and kidneys of mink infected by virus by TUNEL,but no apoptosis was in tongue.In vitro experiments showed that significant inhibition of cell proliferation,accumulation of cells in the G1 phase,and apoptosis in F81 cells infection with MEV.These results indicate that MEV can induce apoptosis in vivo and in vitro.To further determine whether MEV NS1,NS2,VP1 and VP2 can induce apoptosis,we construct of eukaryotic expression vector of NS1,NS2,VP1 and VP2 of MEV,which was transfected into F81 and HEK293 T cells,and analyze apoptosis in theses transfected cells.The results showed that significantly decreased cell viability and significant increase cell lysis in NS1-expressing F81 and HEK293 T cells,while no found in cells expressing VP1 and VP2.The effects of NS1,NS2,VP1 and VP2 on cell cycle and apoptosis were detected by flow cytometry.NS1 induced cell cycle arrest at G1 phase and apoptosis as assayed by cell cycle detection assay and Annexin V-FITC PI assay in both F81 and HEK293 T cells.In order to further find that MEV NS1 could cause chromatin condensation and genomic DNA fragmentation;Electron microscopy shows that chromatin is concentrated,and the nucleus is divided into different sizes and apoptotic bodies are formed.Taken together,our observation strongly suggested only MEV NS1 has the ability to induce apoptosis,while NS2,VP1 and VP2 cannot induce apoptosis.Based on the above finding,further analysis of which apoptosis pathway leads to apoptosis by MEV NS1.The effects of MEV NS1 on intracellular caspase and apoptosis-related proteins were analyzed by Western blot and immunofluorescence.Activation of Caspase-9 and Caspase-3 were subsequently detected,while Caspase-8 and Caspase-12 were not activated.It was also found that NS1 increased the Bax/Bcl-2 ratio,phosphorylated Bcl-2(Ser 70),and promoted the entry of Bax from cytoplasm into mitochondria,and the release of Cyt C mitochondria into cytoplasm.MEV NS1 caused mitochondrial membrane potential depolarization,opening of mitochondrial permeability transition pores and ROS accumulation.NS1 inhibited the activity of p38 MAPK and p53 promoters,and induced the phosphorylation of p38 MAPK(Thr 180/Tyr 182)and p53(Ser 15),p53(Ser 20)and.To further clarify the involving mechanism,Pretreatment with p38 MAPK and p53 specific inhibitors significantly inhibited NS1-induced apoptosis.Taken together,We have reported that MEV NS1 could be able to trigger cell apoptosis through mitochondria-mediated pathway,while p38 MAPK and p53 can play an important regulatory role in NS1 induced apoptosis.