Molecular Mechanism Of Autophagy Induced By Porcine Epidemic Diarrhea Virus In IPEC-J2 Cell

Porcine epidemic diarrhea（PED）is an acute severe infectious disease of the intestine resulting from infection with porcine epidemic diarrhea virus（PEDV）.PEDV has a strong affinity for intestinal epithelial cells,mainly infecting porcine intestinal mucosal epithelial cells where it replicates profusely,causing atrophy and shedding of the intestinal villi.In piglets,the disease is mainly manifested as watery diarrhea,vomiting,and dehydration,with mortality rates approaching100%.However,the pathogenic mechanism is poorly understood.Autophagy is a normal physiological activity that plays an important role in the maintenance of cellular homeostasis.When cells are subjected to stress,such as increased energy demands,autophagic pathways are activated,leading to the lysosomal degradation of aging organelles and the recycling of the breakdown products to maintain optimal cellular function and stability of the intracellular environment.Autophagy is also involved in the clearance of viruses from infected cells.However,there is increasing evidence that many viruses are not cleared by autophagy and utilizing autophagy for their replication.Clarification of the mechanism of autophagy induced by virus infection in cells will lay a theoretical foundation for the in-depth analysis of PEDV pathogenesis and antiviral drug development.In this study,the virulent PEDV CH/HLJ/18 strain was used to infect intestinal porcine epithelial cell line-J2（IPEC-J2）.Firstly,the effects of autophagy on PEDV replication were evaluated,followed by transcriptomic analysis and screening of the cellular receptors involved in PEDV-induced autophagy.The key functional domains and associated signaling pathways of the PEDV non-structural protein 6（Nsp6）in receptor binding and autophagy induction were investigated.Furthermore,changes in the expression of intracellular molecules in IPEC-J2 cells after PEDV infection and their relationships with autophagy were evaluated to elucidate the mechanism by which PEDV induces autophagy and its influence on virus replication.In this study,the TCID₅₀ of the virulent PEDV CH/HLJ/18 strain was measured,and the growth curve of the virus was plotted.After determining the PEDV multiplicity of infection（MOI=1）,autophagy induced by PEDV in IPEC-J2 cells was investigated.Increased levels of the autophagy-associated protein LC3-II were observed 18 h after PEDV infection.Significant increases in the numbers of intracellular double-membrane autophagosomes were also observed in the infected cells,indicative of increased autophagy.N gene copy numbers were detected by qRT-PCR,showing significant increases in the copy numbers in correspondence with the level of autophagy,and indicating that PEDV-induced autophagy was conducive to PEDV replication.PEDV also promoted the accumulation of LC3 and p62 protein in IPEC-J2 cells pretreated with chloroquine,indicating that PEDV induced autophagic flux.Double-fluorescence labeling confirmed the presence of PEDV-induced autophagic flux.To further analyze the role of autophagy in PEDV replication,cells were treated with the autophagy promoter rapamycin and the autophagy inhibitor insulin,followed by the measurement of the PEDV N protein levels.The results showed elevation of N protein levels after rapamycin-induced autophagy,indicating that autophagy promoted PEDV replication.Furthermore,insulin both inhibited autophagy and reduced the N protein levels,indicating that inhibition of autophagy could reduce PEDV replication.To identify the autophagy receptors activated by PEDV in IPEC-J2 cells,the expression patterns of intracellular genes after PEDV infection were analyzed by RNA-seq.This showed that135 genes were up-regulated and 1208 genes were down-regulated 18 h after PEDV infection,compared with uninfected control cells.These differentially expressed genes were analyzed by Gene Ontology（GO）annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analyses.The results showed that the differentially expressed genes were mainly involved in the cell cycle,inflammatory response,apoptosis,autophagy,and metabolism.The most significantly enriched pathways were the steroid biosynthesis,terpenoid biosynthesis,Toll-like receptor signaling pathway,viral proteins and cytokines,and cytokine receptors.Fluorescence-based quantitative PCR was used to verify the expression of randomly selected differentially expressed genes to confirm the accuracy of sequencing.Based on the GO and KEGG enrichment analyses,Toll-like receptor 4（TLR4）,transferrin receptor（TFRC）,andγ-aminobutyric acid type A receptor subunitγ3（GABRG3）were selected as candidate genes for PEDV-induced autophagy.The molecular mechanism of autophagy induced by PEDV in IPEC-J2 cells was further explored.To identify the autophagy-associated receptors involved in PEDV infection of IPEC-J2 cells,the three candidate genes were further investigated by silencing their expression using si-RNA interference.Western blotting showed that while silencing of TLR4 did not affect the LC3-II or p62protein levels in the cells,there was a significant reduction in the level of the viral N protein.This indicated that TLR4 silencing inhibited both PEDV-induced autophagy and PEDV replication.The PEDV N gene copy number was detected by qRT-PCR,and the result was consistent with the protein detection results.To verify that inhibition of TLR4 expression inhibits both autophagy and virus replication,the autophagosome numbers and PEDV N protein levels were measured using an autophagosome detection kit and indirect immunofluorescence assay,confirming that both the numbers of autophagosomes and the fluorescence intensity of the PEDV N protein were significantly reduced after TLR4 silencing.Western blotting also showed that the levels of p-AKT and p-mTOR were significantly increased after TLR4 silencing,while the phosphorylation levels decreased in the control cells,suggesting that autophagy induction occurred through inhibition of AKT and mTOR activity on PEDV infection.Taken together,the results indicate that TLR4 is the key receptor involved in PEDV-induced autophagy,and that activation of the TLR4 receptor can induce autophagy by mediating AKT-mTOR signaling.The PEDV protein Nsp6 is key to the induction of autophagy in host cells.To identify the specific domain on Nsp6 involved in autophagy induction,a eukaryotic expression vector of the truncated Nsp6 gene was constructed and transfected into IPEC-J2 cells,and the autophagy level was detected by western blotting,MDC staining,and dual-fluorescence labeling.The results showed that the functional domain of Nsp61-2C（between amino acids 56-151）increased both LC3-II levels and the numbers of autophagosomes while decreasing p62 levels and inducing autophagic flux,indicating that the Nsp61-2C is responsible for autophagy induction.The co-localization between TLR4 and Nsp61-2C protein was observed in IPEC-J2 cells by indirect immunofluorescence assays.These showed significant reductions in the expression of LC3 Ⅱ and autophagosome numbers in response to decreased TLR4 expression,together with increased phosphorylation of AKT and mTOR,suggesting that Nsp61-2C interacted with TLR4 and induced autophagy through inhibiting the AKT-mTOR signaling pathway.In summary,this study demonstrated that PEDV induced autophagy in IPEC-J2 cells and that PEDV promoted its own replication through autophagy.After screening and analysis,TLR4 was found to be a key gene in PEDV-induced autophagy.The key functional domain of Nsp6 was also identified,and providing a basis for in-depth analysis of the pathogenic mechanism underlying PEDV-mediated autophagy induction and its self-replication in infected cells.The findings also suggest a therapeutic target for the development of antiviral drugs for PEDV infections.