Studies On The Signal Pathway Of Endoplasmic Reticulum Stress Response Induced By DHAV-1 Infection

Duck hepatitis A virus type 1(DHAV-1),belonging to the genus Avihepatovirus in the family Picornaviridae,causes duck viral hepatitis(DHV)in 1~3 weeks ducklings with a lethal rate of 90%.DHAV-1 infection can cause apoptosis in multiple organs of ducklings,especially in liver with a large number of acute apoptosis and liver hemorrhagic necrosis.Therefore,DHAV-1 leads to huge economic losses to the duck industry and restricts seriously the development of duck industry.The endoplasmic reticulum(ER)is an important place for folding and processing of proteins in eukaryotic cells,which is also one of the main sites for virus replication.Virus replication often causes a large number of viral proteins,and ER stress response(ERSR)and unfolded protein response(UPR)will be activated when these proteins can't be correctly folded and output in time.Autophagy exerts a supplementary mechanism similar to ERS,which can degrade misfolded proteins and wastes in cells to maintain a steady state cellular process.ERS plays an important role in many viral infection processes,which is not only related to virus proliferation and cell apoptosis,but also regulates cell autophagy process.In this study,molecular mechanisms of ERS,and its autophagy and cell apoptosis caused by DHAV-1 was studied,which provided theoretical basis for revealing the pathogenesis of DHAV-1 infection.The main contents are as follows:1.Quantitative proteomic analysis of DHAV-1-infected DEF cellsIn this study,tandem mass tag(TMT)quantitative proteomics was used to analyze the protein expression profile of duck embryo fibroblast(DEF)cells infected with DHAV-1 at 48 hours post infection(hpi).Results showed that a total of 5250 proteins were identified,of which 4573 proteins were quantitative.In this study,the threshold of change was 1.3 times and t-test p-value <0.05 was used as the standard of differentially expressed protein(DEPs).Atotal of 507 DEPs were identified,including 171 were up-regulated and 336 were down-regulated.Bioinformatics analysis revealed that DEPs were mainly related to cytoskeleton,immune response,stress response,signal transduction,and energy metabolism.In addition,we found that heat shock proteins(Hsp)such as Hsp family B member 8(Hspb8),immune-related proteins such as tripartite motif containing 25 and melanoma differentiation associated gene 5(MDA5),and activating transcriptional regulatory factor 3(ATF3)were significantly up-regulated,while ribosomal protein families(such as ribosomal protein L35 a,RPL35A)and lysosomal and autophagosome related proteins are mostly down-regulated.Kyoto encyclopedia of genes and genome(KEGG)enrichment analysis showed that lysosomes,autophagosome,and RIG-1-like receptor pathway were significantly enriched.Protein interaction results revealed that ribosomal proteins form a dense interaction network with proteins involved in stress and immunoregulatory.The above results suggested that DHAV-1 infection may cause a large number of proteins or misfolded proteins to accumulate in the ER and induce ERSR,which was accompanied by autophagy and immunity in the process of viral infection.Eight DEPs were selected for parallel reaction monitoring(PRM)verification and Western blot verification.The results showed that the expression levels were consistent with the proteomic analysis results,indicating that the proteomics data in this study are reliable.Based on proteomics data,ERSR was chosen as a starting point to further study the specific signal pathway.2.DHAV-1 infection induces ERSRIn this study,the morphology changes of ER in the process of DHAV-1 infection were observed by transmission electron microscopy(TEM).Electron microscopy results showed that DHAV-1infection can cause obvious ER swelling compared to the mock-infected cells and it was suggested that DHAV-1 infection induced ERSR.The RT-qPCR was used to detect the expression of glucose regulated protein 78(GRP78)at different time points after DHAV-1infection.We found that DHAV-1 infection significantly upregulated the expression level of GRP78 and showed time dependence,confirming that DHAV-1 infection induced ERSR.Western blot results also revealed that the upregulation of GRP78 protein both in DEF cells and BHK-21 cells,indicating that DHAV-1-induced ERSR with no cell-specific.3.Identification of UPR pathway activated by DHAV-1 infectionIn this study,Western blot was used to further determine which arms of UPR were participated the DHAV-1-induced ERS process.Western blot results showed that DHAV-1infection increased the protein expression levels of phosphorylated protein kinase-like ER kinase(PERK),eukaryotic initiation factor-2?(eIF2?),while their total levels were almost no change.Further studies showed that DHAV-1 infection induced the up-regulation of the downstream ATF4,ATF3,the growth arrest and DNA damage-inducible protein(GADD34)and its target protein of the pro-apoptotic protein CHOP.The results indicated that DHAV-1infection activated the PERK pathway.Western blot results showed that inositol-requiring enzyme 1(IRE1)was phosphorylated after DHAV-1 infection.The only substrate of IRE1,X-box binding protein-1(Xbp1)mRNA,was cleaved using RT-PCR method.These results indicated that DHAV-1 infection activated IRE1 pathway.Western blot and RT-qPCR was used to analyze the ATF6 pathway.The results showed that DHAV-1 infection did not promote the degradation of ATF6 precursors,nor did it induce the expression of downstream molecules Calnexin and Calreticulin.These above results suggested that DHAV-1 infection does not activate the ATF6 pathway.Therefore,DHAV-1 infection activated the PERK and IREI pathways of UPR,but not the ATF6 pathway.Western blot results further revealed that multiple encoding proteins of DHAV-1,such as structural proteins VP0,VP1,VP3,and nonstructural proteins 2A2,3C could induce the expression of GRP78 in DEF cells,indicating that structural and nonstructural proteins are involved in ERSR caused by DHAV-1.4.DHAV-1 infection induced ERS-mediated autophagyTo determine whether DHAV-1 infection activated autophagy,we observed the ultrastructure of autophagosomes by transmission electron microscopy(TEM).The results showed that the double-membraned autophagy-like vesicles were generated in the positive control treated with rapamycin(RAPA)and the DHAV-1 infection group,whereas the mock group was not.Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I(LC3-I)to the lipidation form of LC3-II increased with DHAV-1infection.These results suggested that DHAV-1 infection activated autophagy.Notably,the conversion rate of LC3-I to LC3-II was decreased when DEF cells were processed with4-phenylbutyrate(4-PBA).These findings indicated that DHAV-1 infection could cause ERS-induced autophagy in DEF cells,and that ER stress was an important regulatory factor in the activation of autophagy.In order to further study the effects of ERS and autophagy on DHAV-1 replication,cells were treated with Tm,RAPA,4-PBA and DHAV-1,respectively.The RT-qPCR results showed that DHAV-1 mRNA copy number was significantly increased.5.DHAV-1 infection induced ERS-mediated apoptosisIn this study,immunofluorescence and DNA Ladder results showed that DHAV-1infection leads to nucleus shrinkage and DNA rupture,indicating that DHAV-1 infection caused cell apoptosis.Western blot results showed that DHAV-1 infection could induce the??expression of the apoptotic executive molecule of Cleaved-caspase 3,and the expression trend was consistent with the expression of CHOP.Notably,both the expression levels of CHOP and Cleaved-caspase 3 were decreased when cells were treated with 4-PBA.PERK-eIF2?-ATF4-CHOP branch plays a decisive role in ERS-induced apoptosis,and double-stranded-RNA-activated protein kinase(PKR)protein can also promote the expression of p-eIF2?.In this study,cells were treated with ERS inducer tunicamycin,ERS inhibitor4-PBA,PERK inhibitor GSK2606414,and PKR inhibitor 2-AP to study the regulatory effects on DHAV-1-induced apoptosis.Flow cytometry results showed that ERS promoted DHAV-1-induced apoptosis,and inhibition of ERS or PERK/PKR-eIF2?-ATF4 pathway could inhibit DHAV-1-induced apoptosis.Further Western blot and RT-qPCR results showed that inhibition of PERK/PKR-eIF2?-ATF4 pathway inhibited the up-regulation of CHOP and DHAV-1 replication.These results indicated that DHAV-1 infection activated ERS-mediated apoptosis,and the PERK/PKR-eIF2?-ATF4 pathway played an important role in DHAV-1infection.