Mechanism Of SHP-1 Promoting Porcine Epidemic Diarrhea Virus Replication

Porcine Epidemic diarrhea virus(PEDV),a member of the genus Alpha coronavirus,is one of the main pathogens causing diarrhea of piglets.Since the emergence of PEDV variant in 2010,PED outbreaks have occurred repeatedly in China,South Korea,the United States and other countries,causing serious impact on the pig industry.Therefore,it is particularly important to find a way to effectively control PED outbreaks.Innate immunity is the first line of defense against external pathogens.Recognition of pathogene-related molecular patterns(PMAPs)by pattern recognition receptors(PRRs)activates different immune signaling pathways,and finally induces the production of interferon and inflammatory factors,thus inhibiting the replication of pathogens.Currently,several studies have reported that PEDV’s structural and non-structural proteins can inhibit the activation of interferon signaling pathway.In addition,PEDV can promote its own replication by regulating the expression of host proteins.SHP-1 is a protein tyrosine phosphatase,and it has been reported that this protein can promote the replication of VSV,HSV-1 and other viruses by inhibiting the production of interferon.However,there are no reports on the regulation of virus replication by porcine SHP-1.In this study,the effect of SHP-1 on PEDV replication was verified by overexpression and knockdown of SHP-1,and the molecular mechanism of SHP-1 promoting PEDV replication was further explored.This study is mainly divided into the following five parts:1.Construction of eukaryotic expression vectors of RIG-I,MAVS,TRAF3,TBK1,IRF3 in the RIG-I pathway and SHP-1Using PK15 cell c DNA as template,RIG-I,MAVS,TRAF3,TBK1,IRF3 and SHP-1were amplified by PCR for each fragment,the fragments of RIG-I pathway were inserted into p CAGGS-Flag eukaryotic expression vector by homologous recombinant kit,and SHP-1 was inserted into p CAGGS-HA eukaryotic expression vector.After transformation and amplification,the bacterial liquid was taken for PCR amplification,which showed that the vectors were successfully constructed,and the sequence was consistent with expectations.The plasmids were transferred into 293 T cells,and the cells were collected 24 hours later.After lysis and centrifugation,the supernatant was used for western blotting detection.All the proteins with the expected size were observed,which proved that the eukaryotic expression vectors could be successfully expressed in 293 T cells.2.Influence of SHP-1 on PEDV replicationIn order to investigate the effect of SHP-1 on PEDV replication,SHP-1 was firstly transferred into PK15 cells,and 16 h after infection with PEDV virus,the influence of SHP-1on PEDV replication was observed by detecting the m RNA level of virus S protein.The results showed that SHP-1 could promote PEDV replication in a dose-dependent manner.After transfection with si RNA knockdown SHP1,the copy number of PEDV could be reduced,indicating that SHP-1 could promote PEDV replication.3.The influence of SHP-1 on the transcription of IFN-β and three ISGs(TNF-α,ISG15 and CXCL10)To investigate whether SHP-1 promotes PEDV replication by inhibiting the production of interferon,the eukaryotic vector SHP-1 was transferred into PK15 cells and inoculated with Sendai virus.The results showed that overexpression of SHP-1 could reduce the transcription levels of interferon IFN-β and three ISGs(TNF-α,ISG15 and CXCL10)in a dose-dependent manner.After transfection with si RNA knockdown of SHP-1,the transcription levels of IFN-β and the three ISGs increased.It can be proved that the overexpression of SHP-1 can inhibit the production of IFN-β and promote PEDV replication.When SHP-1 is knocked down,the inhibition effect of SHP-1 on interferon is reduced,and the transcription level of interferon is recovered,thus inhibiting PEDV replication.4.Verification of the interaction between SHP-1 and TRAF3In order to find specific proteins in RIG-I pathway interacting with SHP-1,eukaryotic expression vectors of RIG-I,MAVS,TRAF3+TBK1,TBK1 and IRF3 in RIG-I signaling pathway and SHP-1 were transferred into 293 Tcells,respectively.IFN-β-Luc promoter activity and ISRE-Luc promoter activity were detected by double luciferase reporting assay.The results showed that SHP-1 could inhibit the induction of interferon and ISGs by upstream TRAF3 molecules,but had no significant effect on the induction of interferon and ISGs by downstream molecules.It is preliminarily concluded that SHP-1 plays a role by targeting TRAF3.The eukaryotic expression plasmids of TRAF3 and SHP-1 were co-transferred into PK15 cells,and the co-localization of the two proteins was found in PK15 cells by indirect immunofluorescence detection.SHP-1 and TRAF3 were transferred into 293 T cells,and the co-precipitation experiment showed that TRAF3 could precipitate SHP-1,which further demonstrated the interaction between TRAF3 and SHP-1.5.Influence of SHP-1 on ubiquitination of TRAF3In order to explore the specific mechanism of SHP-1’s inhibitory effect,SHP-1,TRAF3 and wild-type ubiquitination plasmid were co-transferred into 293 T cells.Through co-precipitation experiments,it was found that SHP-1 could inhibit the ubiquitination of TRAF3.Further co-transmutation of SHP-1,TRAF3 and K63 ubiquitination plasmids showed that SHP-1 acted by inhibiting K63 ubiquitination of TRAF3.In summary,this study found that SHP-1 can promote PEDV replication,and SHP-1plays a role in promoting PEDV replication mainly by inhibiting K63 ubiquitination of TRAF3 and inhibiting activation of interferon signaling pathway.This study provides a theoretical basis for further revealing the pathogenesis of PEDV and a new idea for scientific prevention and control of PED.