The Effects Of Chloroquine And PEDV On Autophagy At Ultrastructural Level Of Cells

Autophagy is a quite conserve pathway in eukaryotes,responsible to recycle the organelle in cytosol and surrounded by the autophagosomes,then fusion with lysosome to form the autolysosome to degraded it and reused for the next step.While autophagy is relevant to many biological processes and often serves as a gatekeeper of homeostasis,when something goes wrong it can either lead to disease or be activated to fight against it.Moreover,since electron microscope(EM)was first used in 1950s that Christian de Duve found the autophagosome under the EM,scientists use this technic to observe the autophagosomes and to do the quantification of autophagosome under the starvation or the induction of autophagy,thus EM become the golden method to check the autophagy pathway worldwide.Lysosome inhibitors,like Bafilomycin A1(BafA1)and Chloroquine(CQ),have the capacity to inhibitor the fusion step of autophagosome and lysosome.Thus these drugs were used quite often in the experiments and CQ is approved by the Food and Drug Administration(FDA).Therefore,CQ is the mainly compound that currently used in clinical.However,the mechanism of inhibition of autophagy with CQ still needs to be clarified.While chemical drugs have an effect on autophagy,many studies have shown that virus infection can also induce or inhibit autophagy,so as to utilize its related reactions to assist virus replication or reduce the possibility of being degraded by the cell's own metabolic renewal.Porcine Epidemic Diarrhea Virus(PEDV)belongs to the Coronaviridea(CoV)and all the Coronaviruses were grouped into four subgroups,PEDV was in alpha-coronavirus(alpha-CoV).It has a high mortality rate in infected breast-feeding piglets and causing huge losses to agricultural production in the worldwide.Although PEDV is the one of the most serious pathogen for veterinary in recent years,the relationship between PEDV and autophagy is not well investigated and the papers till now for whether PEDV could induce autophagy is still under discussion.At the same time,the membrane rearrangements induced by PEDV Infection still needs to be completed,even by any other alpha-CoVs was still not shown that ultrastructural characterization of membrane rearrangements in a gradual time course.In our studies,we start with ultrastructural analysis of cells expose to these two foreign material via EM.First we use Western blot,long-protein digestion and other methods analyze cell autophagy.After that,we introduced EM to observe ultrastructural characterization induced by drugs or virus infection.Then,with the quantification of immunofluorescence assay and Immuno-EM(IEM),we measure the function of those structure.To further shed light onto the role of membrane rearrangements induced by PEDV infection,in this study we also used time-course to label the virus life cycle of PEDV with a comprehensive time course to determine the structures we observed from which stage of virus,combined with results from viral genome RNA production,viral release and the immunofluorescence results of viral replication.We observed the huge difference of ultrastructural membrane rearrangements under EM,especially for CQ,it induced the similar vesicle structure,comparing to other autolysosome inhibitor and we also observed the large vesicles,likely to have the degraded function,similar to autolysosome.Although both of CQ and BafA1 treatments could block the autophagy flux,we found the morphological changes caused by these two compounds were quite different,so it was possible that the cells and tissues in vitro and in vivo research might be different.Furthermore,we confirmed that treatment of CQ could direct break the fusion of lysosome and autophagosome,whereas BafA1 inhibit the autophagy through effect of the acidity and/or degradation activity of the organelle.Moreover,we concluded that the inhibition of autophagy using CQ treatments was mainly due to the serious confusion of Golgi apparatus and the lysosomal system.Although our western blots results have shown that the lapidated form of LC3 increased with the infection of PEDV,we only could find the autophagosomes,but with a very low frequency.Our results indicated that PEDV infection induced the rearrangements of ER and Golgi before we could observe DMV,convoluted membrane(CM)and large virion-containing vacuole(LVCV).Consistent with other CoV published data,whereas we also found the proliferation of ER and Goligi vacuoles induced the irregular vesicle cluster(IVC)structures.Similar to HCoV-NL63 and TGEV,we found the assembly of PEDV virions mainly took place in Golgi and ER.Therefore,first we use EM to observe ultrastructures and trace flourescence of marker protein,found out many differences between treatment of CQ and BafA1,making relevent research complete.Second,our results showed that PEDV could induce autophagy,and we provided new evidence.Last,the support of the cell inner-membrane structure system is a key factor in the function of the cell.Therefore,it is speculated that PEDV infection-induced membrane structure rearrangement may be an important cause of piglet intestinal dysfunction and diarrhea.In this study,using electron microscopy and fluorescence levels and other related methods to interpret the molecular mechanism of PEDV-induced membrane structure rearrangement biosynthesis may help to more accurately develop new therapies and drugs that inhibit viral replication and open up the in vivo use of these therapies and drugs as an effective preventative and prophylactic treatment.