Identification And Functional Study Of Cell Membrane Proteins Involved In Porcine Epidemic Diarrhea Virus Infection

Porcine epidemic diarrhea virus(PEDV)is an enveloped,positive-stranded RNA virus in the genus Alphacoronavirus.PEDV infection can cause acute intestinal infectious disease,which is characterized watery diarrhea,vomiting and dehydration,and affect pigs of all ages.Since the end of 2010,massive severe outbreaks of PEDV infections have occurred in China,with 80-100% morbidity and 50-90% mortality in suckling piglets,leading to huge economic losses in swine industry worldwide.Enveloped virus particles introduce their genetic material into organisms by binding to cell receptors and then fuse the viral and cell membranes.Membrane proteins play a very important role in the process of virus adsorption,internalization,transportation and release.However,it is not fully understood which host proteins PEDV utilizes,how it enters cells,and the host defense responses.With the development and application of membrane proteomics,the membrane proteins related to virus infection have been identified,which greatly promotes the study of viral pathogenesis.In order to explore the changes of host membrane protein expression when PEDV infected cells,we identified the differentially expressed membrane proteins in Vero cells infected by PEDV variant strain and control cells by isotopic tags for relative and absolute quantification(i TRAQ).Through the analysis and screening of differential proteins,the function and mechanism of PARD3(Partitioning defective 3)and PTPN14(Protein tyrosine phosphatase non-receptor type 14)in PEDV infection were further studied,which laid foundation for revealing the mechanism of PEDV invasion and cell defense.The specific research results are as follows:1.Using recombinantly expressed PEDV-N protein and concentrated and purified PEDV as immunogens,mouse monoclonal antibodies against PEDV N protein and S protein were successfully screened,which laid the foundation for the detection of viral protein expression and viral localization.2.Vero cells infected by HNXX strains can form syncytial lesions.To investigate which membrane proteins were changed during syncytia formation,we successfully identified 21 differentially abundant proteins by extracting cell membrane proteins and using i TRAQ and MALDI-TOF MS/MS,of which 10 were up-regulated and 11 were down-regulated.The differential proteins were classified and analyzed,and it was found that the functions of proteins were related to cell polarity,intracellular material transport,protein phosphatase,histones and acetylation.Among them,12 proteins were identified for the first time in PEDV-infected cells,which provides new clues for the infection and invasion mechanism of PEDV.3.Through analysis and identification of differential proteins,it was found that PARD3 was downregulated after PEDV infection.PARD3 is a PDZ family protein identified in the process of PEDV infection,which is implicated in the formation of tight junctions at epithelial cell-cell contacts.Then,we found that PEDV infection promoted the degradation of PARD3 via the ubiquitin proteasome pathway.Moreover,knockdown of PARD3 promoted the proliferation of PEDV.Further study showed that the downregulation of PARD3 altered the normal morphology of the tight junction proteins and promoted apical and basolateral virus proliferation and promote the apoptosis caused by PEDV.4.PTPN14 is non-receptor protein tyrosine phosphatase which plays an important role in cell adhesion,cell migration and cell growth.It was up-regulated in the results of membrane proteomics.PTPs modulate the cellular level of tyrosine phosphorylation under normal and pathological conditions.In this study,we reported PTPN14 functions as a novel regulator of signal transducer and activator of transcription 3(STAT3)phosphorylation during PEDV infection.Firstly,PTPN14 was markedly upregulated in PEDV-infected Vero cells with the decrease of STAT3 phosphorylation.Knockdown of PTPN14 or phosphatase inhibitor treatment promoted PEDV proliferation and increased the phosphorylation of STAT3 in Vero cells.On the contrary,overexpression of PTPN14 inhibits viral infection in Vero cells.Moreover,dephosphorylation of STAT3 by PTPN14 might occur in the cytoplasm but not in nucleus.Collectively,our results indicate that PTPN14 plays a negative role in regulating STAT3 activation in PEDV infected Vero cells and demonstrate another layer of regulation in PEDV infection.In conclusion,we identified the differentially expressed membrane proteins in PEDV infected Vero cells for the first time.Through the further study of PARD3 and PTPN14,we revealed the mechanism and influence of cell polarity and phosphatase in PEDV infection for the first time,so as to provide a new scientific basis for the study of PEDV invasion and host defense mechanism.