Molecular Mechanism Of Autophagy Mediated By Endoplasmic Reticulum Stress Induced By Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)is the causative agent of porcine epidemic diarrhea(PED),an acute and highly contagious enteric viral disease.PEDV infects swine of all ages and causes symptoms such as vomiting and diarrhea.PEDV only produced mild symptoms such as emaciation when infected with adult pigs,while it showed a mortality rate of nearly 100%when infected with newborn piglets.The disease is still endemic in many countries and regions since it was first reported in the United Kingdom in 1971.Moreover,the mutation of the endemic strain of PEDV and the lack of an effective vaccine have caused significant economic losses to the world pig industry.This project investigates the regulatory mechanism of PEDV on Vero cell autophagy and the effects of autophagy on the proliferation were analyzed to provide theoretical support for revealing the molecular pathogenesis of PEDV and the prevention and control of PEDV.In order to verify whether ER stress is activated in PEDV-infected Vero cells.Vero cells were infected with PEDV(MOI of 0.5)and collected at 0,6,12,18 and 24 h post-infection.The expression level of GRP78 is detected.Western Blot shows that PEDV infection increased the expression of GRP78,which suggests that PEDV infection induced ER stress.Western blot was used to detect the activation level of PERK/e IF2,IRE1/JNK and ATF6signal pathways in PEDV-infected cells.The results show that PEDV infection respectively up-regulated the phosphorylation levels of PERK,e IF2a,IREI,and JNK,up-regulated the expression of the cleaved ATF6 protein.These results indicate PEDV infection activated PERK/e IF2/,IREI/JNK and ATF6 signaling pathways.To determine whether PEDV infection induces autophagy,the expression level of LC3?protein and p62 protein were detected by western blot.The results showed that PEDV infection increased the expression of LC3II/LC3?and dereased expression of p62 protein.These results suggest that PEDV infection can induce autophagy.ER stress is one of the causes of autophagy.To determine whether PEDV infection-induced ER stress promotes autophagy,cells were treated with 4-phenylbutyrate(4-PBA)for 1 h before infection with PEDV,and the level of autophagy was detected by western blot.The results showed that,compared with PEDV-infected cells,4-PBA decreased the ratio of LC3II/LC3I,which indicated that PEDV infection induced autophagy through ER stress in Vero cells.Further study was adopted to verify whether the UPR signaling pathway is involved in the autophagy process induced by PEDV,PERK inhibitor GSK2606414,IREI inhibitor STF-083010 and ATF6 inhibitor AEBSF were respectively used to inhibit PERK,IRE1 and ATF6 signaling pathways,and the level of autophagy was detected by western blot.The results showed that,compared with PEDV-infected cells,GSK2606414,STF-083010 but not ATF6 inhibitor AEBSF decreased the ratio of LC3II/LC3I,which indicated that PERK and IRE1 signaling pathways participated in the regulation of autophagy.To explore the relationship between autophagy and viral replication in Vero cells infected with PEDV.Autophagy inducer rapamycin and autophagy inhibitor3-Methyladenine(3MA)as controls,PERK inhibitor GSK2606414 and IREI inhibitor STF-083010 were exposed to Vero cells,and virus replication was detected by western blot,RT-q PCR and TCID₅₀.The results showed that rapamycin facilitate viral replication,while3-MA,GSK2606414 and STF-083010 treatment decreased viral replication.The results showed that PEDV infection activated autophagy to facilitate viral replication through PERK and IRE1 signaling pathways.Reactive oxygen species(ROS)plays an important role in the regulation of multiple signaling pathways.N-acetylcysteine(NAC),a reactive oxygen scavenger,was used to explore the role of ROS in PEDV infestation-induced ER stress and autophagy.Western blot analysis showed that NAC treatment decreased GRP78 expression and LC3II/LC3I protein expression ratio compared with PEDV-infected cells,indicating that ROS acts as an upstream regulator of ER stress and autophagy.In summary,these results provide the mechanism of PEDV-induced ROS-dependent ER stress-mediated autophagy in Vero cells through activating the PERK/e IF2 and IRE1/JNK pathways.