The Molecular Mechanisms Of Autophagy Induced By VP1 Protein Of Duck Hepatitis A Virus Type 1

Duck Hepatitis A Virus Type 1(DHAV-1)belongs to the Picornavirus family and the genus Avian Hepatitis.Duck viral hepatitis(DVH)caused by DHAV-1 infection is a highly lethal infectious disease that has caused huge losses to the duck industry worldwide.Autophagy is an effective intracellular decomposition pathway that relies on lysosomes.It is used to degrade abnormal cellular protein aggregates and damaged organelles.It has a high degree of conservation in the evolutionary process and is effective in maintaining the stability of the intracellular environment,playing a key role in the state process.Although autophagy was originally thought to be a cellular stress response,more and more evidence shows that it plays a normal physiological role in a variety of biological processes.Studies have shown that autophagy is involved in the regulation of many diseases,and the regulation of autophagy is also affected by many signaling pathway molecules.The endoplasmic reticulum(ER)is an important membranous organelle,which exists in eukaryotic cells and plays a key role in lipid biosynthesis,calcium storage and glucose metabolism.As a site for cell protein modification and folding,ER is also an essential organelle in the process of virus replication and maturation.In the infecting process,the large amount of synthesized viral protein in the ER usually leads to the accumulation of protein,causing endoplasmic reticulum stress(ERS).To eliminate the accumulated protein in the ER,the cell would initiate an unfolded protein response(UPR)and restores its homeostasis.Although it has been reported that active DHAV-1 could induce ERS-mediated autophagy,it is still unclear whether its main capsid protein VP1 plays an important role in the molecular mechanism of DHAV-1 inducing ERS-mediated autophagy.Therefore,this study mainly focuses on the molecular mechanism of ERS induced by DHAV-1 VP1 protein and its autophagy-mediated autophagy.The research content is mainly divided into the following three parts:1.Eukaryotic expression of DHAV-1 VP1 proteinUsing the DHAV-1 infectious clone as a template,the VP1 gene fragment was amplified by PCR,and cloned into the eukaryotic expression vector pc DNA3.1 through the Bam H I and Xho I restriction sites,and then double-enzyme digestion was performed to identify the recombinant plasmid.The recombinant plasmid was transiently transfected into DEF cells.After 24 hours of treatment,a protein band with a molecular weight of about 47 k Da was detected by Western blotting.The DEF cells transfected with the empty vector and recombinant plasmid were fixed and nucleated.It was observed by laser confocal microscope that there was no green fluorescence in the cells in the empty vector group,but green fluorescence in the cells in the plasmid transfected group,indicating that the VP1 protein was successfully expressed in DEF cells.2.VP1 protein induces autophagy in DEF cellsTo investigate whether DHAV-1 VP1 protein induces autophagy,we transfected GFP-LC3 plasmid into DEF cells.Observed by laser confocal microscopy,it was found that expression of VP1 protein can induce the accumulation of autophagosomes.Western blotting detection revealed that VP1 protein was expressed.It can induce the transformation of LC3-I to LC3-II.The results show that the VP1 protein can induce autophagy in DEF cells,and it is time dependent.VP1 protein was expressed in BHK cells.Western blotting detection revealed that VP1 protein expression can also induce the conversion of LC3-I to LC3-II,indicating that autophagy induced by VP1 protein is not cell-specific.In addition,in order to explore whether the autophagy induced by VP1 protein is complete,we tested the expression level of p62,the co-localization of m RFP-GFP-LC3,and the expression levels of p62 and LC3-II after adding CQ.The results showed that the fusion of autophagy and lysosome was blocked by VP1 protein transfection,that is,VP1 protein did not induce the production of complete autophagy flow.3.Research on the signal pathway of VP1 protein inducing autophagyFirstly,the expression of glucose-regulated protein 78(GRP78)at different time points after the expression of VP1 protein was detected by RT-q PCR and Western blotting.The results showed that the expression of GRP78 began to increase after VP1 transfection into DEF cells,indicating that VP1 protein can induce ERS in DEF cells in the early stage;in addition,we detected the expression levels of p-PERK,p-e IF2?,and ATF4.The results showed that the expression levels of all three were increased compared with the blank group,indicating that the VP1 protein can activate the PERK-e IF2?-ATF4 signaling pathway.Subsequently,it was found that treatment of cells with 4-PBA significantly inhibited the conversion of LC3-I to LC3-II,which preliminarily indicated that autophagy induced by VP1 protein is regulated by ERS.Finally,we treated DEF cells with PERK-specific inhibitors and found that the inhibitors significantly inhibited the expression of p-e IF2? and LC3-II after treating DEF cells,indicating that VP1 protein induced autophagy by activating the PERK/e IF2? pathway.In summary,this study found that DHAV-1 VP1 protein could activate ERS through the PERK-e IF2?-ATF4 signaling pathway,thereby inducing autophagy.This study provides a theoretical basis for further revealing the pathogenic mechanism of DHAV-1 infection and provides a new idea for the scientific prevention and control of DVH.