Mechanisms Of Porcine Epidemic Diarrhea Virus-induced Autophagy

Background:Autophagy is a highly conserved self-digestion process that plays a crucial role in maintaining cellular homeostasis in response to nutrient depletion or other cellular stresses such as accumulation of damaged organelles,unneeded protein aggregates,and invading microbes.Autophagy is controled by mTOR and AMPK,two nutrient-and energy-sensitive kinases.These two kinases phosphorylate ULK1/2 at different serine residues and have the opposite effect on ULK activity:mTOR phosphorylates ULK1 at serine 757(ULK1S757)and inhibits its activity;whereas AMPK phosphorylates ULK1 at multiple sites,including the serine residues 317,555,and activates its activity.ULK1/2 binds ATG13 and FIP200 proteins to form a preinitiation complex,which controls the activation of the initiation complex that comprises Beclin 1,ATG14L,VPS34,and VSP15.VPS34 is a Class ? PI-3 kinase and catalyzes phosphatidylinositol(PI)-4,5 to PI 3-phosphate(PI3P),which initiates the elongation and nucleation of the double membrane to form autophagosomes.The autophagy pathway is an essential component of host defense system against viral infection.Porcine epidemic diarrhea(PED)is a devastating enteric disease,caused by porcine epidemic diarrhea virus(PEDV).PED characterized by severe water diarrhea,vomiting and dehydration in infected swine.PEDV is a single-stranded,positive-sense RNA virus,belonging to the family Coronaviridae,subfamily Coronavirinae.Our present study focuses on the ability of PEDV to induces autophagy and to understand its underlying molecular mechanism if it does.Methods:Western blot was used to detect the levels of LC3 and p62 in Vero cells infected with various amounts of the multiplicity of infection(MOI)of PEDV for the indicated lengths of time.Confocal microscopy was used to determine the ability of PEDV to induce the formation of autophagosome and autolysosomes.Whether PEDV induced functional autophagy was determined by adding bafilomycin A1 and chloroquine(CQ)into Vero cells in PEDV-infected cells.TCIDso was used to measure virus titer in the cell lysates the conditioned media.Results:PEDV increased the levels of LC3-II and p62 levels in a time-and dose-dependent manner.Bafilomycin A1 and CQ both increased the levels of LC3-II but blocked the change of p62 expression.PEDV infection also induced the formation of autophagosome and autolysosome,as evidence by the presence of red puncta in GFP-RFP-LC3-expressed Vero cells.These observations collectively suggest that PEDV induces functional autophagy.These inhibitors also reduced the expression of viral proteins and lowered the virus titer in the conditioned media,indicating that PEDV-induced autophagy can promote PEDV replication.We next investigated the signaling pathway involved in PEDV-induced autophagy.Western blot revealed that PEDV infection did not affect the PI-3 kinase pathway since there was no significant change in the levels of S6K1,S6,and mTOR phosphorylation.These observations suggest that PEDV-induced autophagy is not mediated by the suppression of mTOR activity.In contrast,we found that PEDV dramatically induced AMPK phosphorylation in a time-and dose-dependent manner.Intriguingly,ULK1S317 and ULK1S555 phosphorylation was not increased but rather decreased.Further studies showed that inhibition of ULK1 activity by its specific inhibitor SBI-0206965 led to the suppression of autophagy and PEDV replication.Finally,we investigated the role of JNK activation in PEDV-induced autophagy and virus replication.We found that PEDV induced JNK and Jun phosphorylation in a time-and dose-dependent manner.Inhibition of JNK activity by its specific inhibitor SP600125 led to the suppression of autophagy and PEDV replicationConclusions:PEDV infection induces autophagy by activating JNK1 and ULK1 through AMPK in Vero cells.Further study suggests that autophagy facilitates PEDV replication.