The Study On Autophagy Induced By PHEV Protein PLP In N2a Cells

Porcine hemagglutinating encephalomyelitis(PHE)caused by PHEV is an acute and highly contagious disease,which led to huge economic losses to the pig industry in China.PHEV has the typical characteristics of neurotropic coronavirus,and the nerve cells in brain tissue are the main site of the virus replication.Generally,invasion of virus to the cells will stimulate the immune response of cells,this could help to clear the exogenous pathogen and maintain the cell homeostasis.Autophagy is such a kind of immune mechanism,by which pathogen coated endosomes can be delivered to the lysosome and then inhibit the replication of the pathogens.However,people have found that virus has evolved some regulatory mechanisms to fight with the host cells autophagy,thus provide favorable conditions for the replication.Recent studies have reported that PHEV is able to induce incomplete autophagy,providing favorable conditions for their survival.Papain-like protease(PLP)is an important protease during virus replication.Studies have shown that coronaviruses such as SARS,PEDV,HCoV,and MERS can regulate the formation of host autophagosome through PLP,causing incomplete autophagy.The phages negatively regulate the antiviral immune response.PHEV belongs to the coronavirus and whether PLP plays a role in the process of PHEV-induced autophagy attracted our attention.In this study,we mainly focus on PLP and we would like to know whether PLP could cause autophagy.We constructed an eukaryotic expression vector of pCMV-Flag-PLP,after which PLP was overexpressed in N2 a cells.Firstly,the expression of LC3 protein was detected by the western blot analysis after plasmid pCMV-Flag-PLP was transfected into N2 a cells,and the results showed that the conversion of LC3-I to LC3-II was largely enhanced,indicating the possibility of autophagy.Subsequently,by co-transfecting GFP-LC3 B and pCMV-Flag-PLP plasmids,we have found the accumulation of green fluorescent spots of GFP-LC3 after transfection of PLP-transfected cells was observed,and this process appeared time dependent,further confirming that PLP can induce autophagy in the cells.At the same time,the autophagic substrate protein p62 and lysosomal marker protein LAMP1 was further examined by western blot,and we have found that compared with the control group,the expression level did not change significantly,but occurred retention,suggesting that PLP induced incomplete autophagy in N2 a cells.Previous studies showed that Beclin1 is an important autophagy related protein,which plays important role during the formation and maturation of autophagosome,so here we focus on Beclin1 in the context of PLP-induced autophagy.Firstly,Beclin1 expression was detected by the western blot analysis after PLP overexpression in N2 a cells,the results have shown that Beclin1 was upregulated when compared with the control group,which indicate that Beclin1 is involved in the process of PLP-induced autophagy.It was reported that many kinds of viruses interact with Beclin1 by harnessing their own proteins,which may block the process of fusion between autophagosome and lysosome,providing conditions for virus survival.Secondly,an eukaryotic expression vector of Beclin1 was constructed as pCMV-Flag-Beclin1 and this plasmid was co-transfected with pEGFP-PHEV-PLP into 293 T cells in order to overespress Beclin1 and PLP proteins,and then co-immunoprecipitation experiments were carried out.The results showed that the interaction between PLP and Beclin1 exists.To further verify the above conclusions,immunofluorescence analysis was used to detect the localization of Beclin1 and PLP.And we found that Beclin1 and PLP were obviously colocalized in cells,suggesting that PLP also adopts a strategy similar to that of the above viruses to inhibit Beclin1 interaction.The fusion process of phages and lysosomes provides favorable conditions for their own survival.However,the specific mechanism still needs further study.As an important autophagy-related protein,Beclin1 is involved in regulation of PLP-induced autophagy.However,whether Beclin1 plays a key role in PHEV infection is unclear.In order to study the effect of Beclin1 on PHEV proliferation,the expression level of Beclin1 was detected by quantitative PCR and western blot analysis at indicated time points post PHEV infection.Compared with the control group,Beclin1 showed a clear up-regulation after PHEV infection.To further clarify the role of Beclin1 in PHEV replication,immunofluorescence analysis was used to detect the localization of Beclin1 and PHEV,and we found that Beclin1 and PHEV were obviously colocalized in N2 a cells.Subsequently,an eukaryotic expression vector of Beclin1 was constructed as pCMV-Flag-Beclin1.After transfection into N2 a cells,PHEV was inoculated and found PHEV proliferation had no significant change compared with the control group.RNA interference was used to knock down of Beclin1 in N2 a cells and then inoculated with PHEV.We found the expression of PHEV was significantly down-regulated.In summary,PHEV coded PLP induces incomplete autophagy in N2 a cells,and PLP also induces the expression of Beclin1 protein and at the same time interacts with Beclin1,we further confirm that Beclin1 is necessary for PHEV replication.So here we speculate that PHEV may replicate in the cells through the incomplete autophagy induced by PLP-Beclin1 interaction.Our study will provide new indications for uncovering PHEV caused disease.