Regulation Of Non-Structural Proteins In Japanese Encephalitis Virus On Endoplasmic Reticulum Stressautophagy

Japanese encephalitis(JE)is an acute and severe zoonotic infectious disease caused by Japanese encephalitis virus(JEV),which causes encephalitis of human beings and reproductive disorders of pigs,and brings great harm to public health safety and pig industry.JEV infection can cause endoplasmic reticulum stress and autophagy.After virus infection,the endoplasmic reticulum shows obvious proliferation and hypertrophy.The host endoplasmic reticulum proteins begin to prevent viral RNA translation,degrade misfolded proteins,and activate endoplasmic reticulum stress response through UPR signaling pathway.Endoplasmic reticulum stress can induce autophagy.Autophagy,as a complementary mechanism similar to endoplasmic reticulum stress,maintains cellular homeostasis by regulating immune function.Autophagy lysosome is the main way to degrade autophagy and inhibit virus infection.Endoplasmic reticulum stress and moderate activation of autophagy play an important role in eliminating foreign pathogens and inhibiting virus proliferation.In the early stage of JEV infection,non coding proteins play a role in viral replication,while the regulation of endoplasmic reticulum stress autophagy by non coding proteins of JEV has not been reported.In order to explore the regulatory mechanism of viral response to endoplasmic reticulum stress autophagy,we studied the regulatory effects of viral non coding proteins on endoplasmic reticulum stress and autophagy respectively,in order to obtain the main viral coding proteins that can regulate these two responses,so as to further clarify the correlation mechanism between endoplasmic reticulum stress and autophagy and screen new drug targets.The specific work is as follows:1.Effects of JEV infection on endoplasmic reticulum stress and autophagy in different host cellsIn this study,JEV was used to infect BV2 cells and neuro-2 cells respectively ? BV2 cells were observed for 6h,Neuro-2a and BHK21 cells were observed for 24 h.The samples were collected and detected by real-time PCR and Western blot.The results showed that JEV could induce the increase of GRP78,ATF4 mRNA levels,the phosphorylation of peif2 a protein,the accumulation of sXBP1 mRNA,and the activation of perk and IRE1 pathways in BV2 cells;ATF6 did not form splicing body and could not activate ATF6 signaling pathway.Neuro-2 after JEV infection ? GRP78 mRNA and protein levels were significantly increased in BHK21 and BHK21 cells,which could activate endoplasmic reticulum stress response.At the same time,the positive control group of rapamycin treated BV2 cells for 6 hours was established.Real time PCR and Western blot analysis showed that JEV could induce the autophagy related proteins such as ATG5,Atg7,Beclin1,Rab9,ulk1 and vps34 to increase significantly,and promote the transformation of LC3 ? to LC3 ?;The mRNA levels of ATG5,Atg7,Beclin1 and other autophagy factors were significantly increased.JEV could activate the autophagy of BV2 cells.In order to study the relationship between JEV activated endoplasmic reticulum stress and autophagy,4-phenylbutyric acid(4-PBA)was added to JEV infected cells 2 hours after infection.After 4 hours,GRP78 mRNA and protein expression,and the transformation of LC3 ?to LC3 ? were detected,The autophagy induced by JEV is related to endoplasmic reticulum stress.2.Effect of JEV nonstructural protein on endoplasmic reticulum stress in BV2 cells The constructed JEV fusion protein was transiently transfected into BV2 cells.After 12 hours,the protein samples were collected and detected by Western blot.The results showed that the expression of JEV nonstructural protein except NS1 could significantly increase GRP78 protein level and activate endoplasmic reticulum stress response.NS2 B,NS3,NS4 A,NS4B and NS5 B phosphorylate p EIF2? protein and activate perk pathway.The ATF6 pathway could not be activated without ATF6 splice.Non structural proteins of JEV can accumulate sXBP1 mRNA and activate IRE1 signaling pathway,and NS3 can significantly accumulate sXBP1.3.Effect of JEV nonstructural protein on autophagy pathway of BV2 cells JEV eukaryotic expression vector was transiently transfected into BV2 cells.After 12 hours,the protein samples were collected,and the esterification of autophagy related protein LC3 was detected by Western blot.Three JEV fusion proteins,NS3,NS4 B and NS5 B,significantly increased the expression of LC3? and LC3?,which activated autophagy.Confocal laser scanning showed that NS3 could significantly aggregate exogenous LC3 ? in the cytoplasm,which might activate autophagy through IRE1 pathway.These results demonstrated that JEV nonstructural proteins mainly affect BV2 cells to activate endoplasmic reticulum stress through perk pathway and ire pathway.NS3,NS4 B and NS5 B nonstructural proteins screened are the key proteins that associate endoplasmic reticulum stress with autophagy and participate in JEV mediated innate immunity.The results provide clues for the analysis of the interaction between JEV and host cells,open up new ideas for the study of JEV molecular pathogenesis,and provide drug targets for the treatment of Japanese encephalitis.