Studies On Enterovirus71 Viral Proteins Involves In Cell Autophagy

AIM:We first investigated whether EV71 virus can induce autophagy,and then furth er screened out the EV71 virus protein that can cause autophagy.Methods: The specific content is divided into two chapters.In the first chapter,the changes of LC3 and P62 in EV71 infected cells will be detected by EV71(Hubei Xiangfan strain,JQ804832)and 293 T.In The second chapter,11 kinds of recombinant eukaryotic expression plasmid will be consutrcted by genetic engineering,which were pc DNA3.1(+)-HA-VP1,pc DNA3.1(+)-HA-VP2,pcDNA3.1(+)-HA-VP3,pc DNA3.1(+)-HA-VP4,pc DNA3.1(+)-HA-2A,pc DNA3.1(+)-HA-2B,pcDNA3.1(+)-HA-2C(+),-HA-3A,pc DNA3.1 pcDNA3.1(+)-HA-3AB,pcDNA3.1(+)-HA-3C,pc DNA3.1(+)-HA-3D.After these lasmides were verified by enzyme digestion and sequencing identification,the recombinant plasmid with autophagy marker(pEGFP-LC3)will be transfected into 293 T cells.The protein of the recombinant virus(HAtag)will be dyed by immunohistochemistry.Green punctate fluorescence accumulation and counting will be observed by fluorescence microscopy;observation of colocalization of EV71 LC3 will be used by immunofluorescence confocal microscopy.Result: In EV71 infected 293 T cells which were transfected with pEGFP-LC3 plasmid,we can see a bit of green fluorescence,and both of them show Colocalization.The expression of P62 protein decreased,and the ratio of LC3-?/LC3-was increased.In 293 T cells,which were cotransfected with 11 kinds of viral proteins such as pc DNA3.1(+)-HA-VP1 and pEGFP-LC3,a bright green fluorescence can be seen in the cells with transfection of VP1 and 2A and Western blot flake detected that the LC3-?/LC3-? ratio was increased,p62 protein level was decreased.Conclusion:EV71 virus can induce autophagy and the viral structural protein VP1 and nonstructural protein 2A can induce autophagy.