Proteomics Of PEDV-infected Porcine Jejunum Tissues And Molecular Mechanisms For The Effect Of HnRNP A1 On PEDV Replication

Recently,due to the emergence of porcine epidemic diarrhea virus(PEDV)variant strains,new outbreak of porcine epidemic diarrhea(PED)has caused enormous economic losses to world pig industry.However,it is difficult to deal with the threat from PED because of the lack of prevention and control methods.Therefore,it is meaningful to study the pathogenic mechanism of PEDV and invent new anti-PEDV drugs in terms of controlling and treating this disease.In this study,an iTRAQ-based comparative proteomic analysis was performed to investigate the proteomic changes in the jejunum tissues infected with a virulent PEDV strain and its attenuated strain,which would make for revealing the pathogenic mechanism of PEDV and how the host immune system responses to PEDV infection.According to the results of proteomics,we put emphasis on the interaction between heterogeneous nuclear ribonucleoprotein A1(hn RNP A1)and PEDV N protein and make a breakthrough which can help us understand the way of PEDV replication.This research also investigated the anti-PEDV effect of quercetin and clarified the antiviral mechanism,which could provide a reference for developing related treatments and medicines.The specific research content is as follows: 1.Comparative Proteome Analysis of Porcine Jejunum Tissues in Response to a Virulent Strain of Porcine Epidemic Diarrhea Virus and Its Attenuated Strain A comparative proteomic analysis was carried out to investigate the proteomic changes produced in the primary target organ,using isobaric tags for relative and absolute quantitation(iTRAQ)labeling,followed by liquid chromatography tandem-mass spectrometry(LC-MS/MS).A total of 269 and 301 differently expressed proteins(DEPs)were identified in the jejunum tissues of the piglets inoculated YN13 strain and YN144 strain.Bioinformatics analysis revealed that these proteins were involved in stress responses,signal transduction,and the immune system.By further analysis,many interferon-stimulated genes were found to be up-regulated,indicating that JAK-STAT signaling pathway could be activated by PEDV infection in vivo.Based on the comparative analysis,we proposed that different changes induced by YN13 and YN144 in some transcription and translation related factors,and some members in the heat shock protein(HSP)family,may be responsible for the differences in their pathogenicity.2.hnRNP A1 and PEDV replicationAccording to the result of our proteomic study,hnRNP A1 was found to be differently regulated between YN13 infected group and YN144 infected group.Therefore,we speculated this protein may play a role in the pathogenicity of PEDV.Firstly,colocalization of PEDV N protein and hnRNP A1 was found in PEDV infected Vero cells by Laser co-focus light microscopy,indicating that these two proteins cloud interact with each other in Vero cells.Through co-immunoprecipitation,interaction between hn RNP A1 and PEDV N protein was further confirmed.According to the result of confocal microscopy analysis,hnRNP A1 was found to colocalize with N protein in the perinuclear region,in which active coronavirus replication/transcription complexes reside.Therefore,hnRNP A1 may participate in constituting the PEDV replication/transcription complex and influence PEDV replication.In order to test the participation of hnRNP A1 in PEDV replication,Vero cells were transfected with siRNAs against hnRNP A1 and then were infected with different PEDV strains.The results of RT-PCR,IFA and western blot showed that silencing hnRNP A1 can lead to the reduction of PEDV's genome,the number of PEDV infected cells and expression of PEDV N protein.This revealed that hnRNP A1 plays a positive role in PEDV replication.Combining with our proteomics study,downregulation of hnRNP A1 may be one of the reasons responsible for the lower pathogenicity of YN144 than YN13 in vivo.3.Mechanism of the anti-PEDV activity of quercetin Based on results of indirect immunofluorescence assay(IFA)and western blot,quercetin was found to be with excellent ant-PEDV effect.Firstly,we studied whether quercetin could influence the attachment and invasion of PEDV.The result showed quercetin did not play a function in these aspects.Therefore,quercetin must inhibit PEDV within cells.Since quercetin is a good inhibitor for HSPA1,it may suppress PEDV by inhibiting HSPA1 expression.To testify it,Vero cells were transfected with siRNA against HSPA1 and then were infected with PEDV.The results of RT-PCR,IFA and western blot indicated that inhibiting HSPA1 has no influence on PEDV infection.It meant that anti-PEDV effect of quercetin did not depend on its activity of HSPA1 inhibitor.Through a docking analysis,quercetin was found to be able to bind the active site of PEDV 3C-like protease,indicating that quercetin might interact with PEDV 3C like protease and inhibit its activity.In order to test it,PEDV 3C-like protease was expressed and purified and then was proved to be able to interact with quercetin by using SPR analysis.In addition,a fluorescence resonance energy transfer(FRET)method was established to testify whether quercetin have a negative effect on the activity of PEDV 3C-like protease.Firstly,we tested the activity of our purified proteins in the FRET assay and proved that they were with high activity.However,when quercetin was added in the reaction,activity of our purified proteins was inhibited.Therefore,we considered that quercetin could inhibit PEDV 3C-like protease and then suppress PEDV infection in vitro.