The Function Of Snapin On Nerve Cells Autophagy Induced By Porcine Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis virus(PHEV)is a member of the beta-coronavirus and is one of the major pathogens causing typical encephalomyelitis symptoms in clinically infected pigs.PHEV could mainly infect piglets,especially the sucking piglets,and the mortality rate of up to 20~100%.After PHEV infection,the nerve damage is the primary characteristic.In the brain tissue,nerve cells are the site of the virus replication,no evidence of infection in glial cells.In general,the invasion to cells by virus will stimulate the immune response of cells,aim to clear the pathogen of exogenous invasion and maintain the cell homeostasis.But the virus can also use some components of its cells to provide raw materials for replication,resulting in the body damage.Autophagy is an important process that plays a role in pathogens against cells.It has been confirmed that autophagy is closely related to tumor,neurodegenerative diseases,metabolic diseases,immune diseases and other pathogenesis.A variety of viruses,bacteria and other pathogens infected cells have induce the autophagy or its inhibition.As previously mentioned,PHEV mainly infected nerve cells,whether the process of infection could induce autophagy has not been reported yet.Therefore,in order to clarify whether PHEV could induce autophagy in neurons,we have studied and analyzed the process through a series of autophagic related experimental.N2 a cells with PHEV infection were observed by transmission electron microscopy(TEM),and there were typical bilayer or monolayer structure of autophagosomes in the cytoplasm.In addition,using indirect/direct immunofluorescence and Western blot,it showed that PHEV could promote the formation of GFP-LC3 in N2 a cells and promote the transformation of LC3,indicating that PHEV infection can induce autophagy in neurons.However,during the process of PHEV infection,the autophagic substrate protein p62 protein did not degrade,while the use of chloroquine(CQ)treatment,PHEV infection was significantly reduced.Subsequently,further studies have been carried out on the degradation of autophagosomes.Using tandem-linked fluorescent reporter gene mRFP-GFP-LC3(ptfLC3),and acidic late endosomes and lysosomal markers LysoTracker,we found that the autophagy was not fused to lysosomes in the PHEV-infected group,and the ptfLC3-fluorescent reporter plasmid showed no quenching,and GFP-LC3 and LysoTracker were not co-located in PHEV-infected cells.At the same time,the analysis of lysosome-associated membrane protein 1(LAMP1)showed that PHEV infection caused the abnormal accumulation of LAMP1 and increased by the PHEV infection,it further indicated that PHEV inhibit fusion between the autophagy and lysosomes.According to the results above,it suggested that PHEV induced autophagy which was an incomplete autophagical effect.In order to further clarify the mechanism of autophagy induced by PHEV in N2 a cells,we used yeast two-hybrid system to screen the interaction proteins with PHEV S1 protein.After the preliminary screening and sequencing,we obtained 14 candidate genes.By the analysis of the results,it was confirmed that Snapin protein may be involved in the process of PHEV induced autophagy.Snapin,which is a new type of small molecule protein,belongings to a member of the SNARE complex,which not only plays a transport function in the vesicular transport mechanism,and plays an important role in the process of the degradation of late endosomes and the formation of the autophagosomes.We first verify the interaction between the PHEV S1 and Snapin using the turnover assay on yeast two-hybrid,immunoprecipitation and indirect immunofluorescence.After overexpression of Snapin protein with PHEV infection,the replication did not change significantly,but under the treatment of siRNA interference to Snapin with PHEV infection,the replication was apparently inhibited.At the same time,the detection of lysosomal markers showed that the accumulation of autophagosomes increased significantly compared to the control samples.The results further confirmed that PHEV induced autophagy during infection in N2 a cells,and Snapin was involved in the process and was likely to be associated with the incomplete autophagy induced by PHEV.These results not only provide a basis for the study of mechanisms towards the nerve injury induced by PHEV,but also provide an important theoretical basis for neurodegenerative disease model.