Construction Of Transcription Factor EP0 Gene Deletion Strain Of Pseudorabies Virus And Screening Interaction Proteins Of EP0

Porcine pseudorabies is an acute infectious disease caused by pseudorabies virus(PRV),which can cause breeding pig reproductive disorders,high mortality in nursing piglets and stunted growth of fattening pigs,which can cause huge economic losses to the pig industry.PRV is a double-stranded DNA virus whose genome encodes more than 70 proteins,and the expression of these proteins is cascading temporally.The PRV early expression protein 0(EP0)has the activity of ubiquitinated ligase and can trans-regulate viral gene expression,but its mechanism of regulating PRV transcription is not clear.Here,based on the galk forward and reverse screening system,EP0-deficient PRV strains construction,bioinformatics analysis,LC-MS/MS protein profiling,Western blot and other methods were used to screen host proteins interacting with EP0 proteins.Our results will provide a foundation for further study on EP0 transcriptional regulation mechanism.In this study,the galk positive screening method was used to construct EP0 gene deletion strains and HA-labeled EP0 recombinant positive clonal bacteria SW102-BAC-DEP0 and SW102-BAC-HA-EP0 based on the laboratory-preserved p BAC-JS2012 infectious clone.The BAC recombinant vectors of the above positive bacteria were extracted PRV-BACDEP0 and PRV-BAC-HA-EP0 were co-transfected with the cre enzyme expression vector pc DNA3.1-cre to Vero cells,and the EP0-deficient virus strain PRV-DEP0 and recombinant virus strain PRV-HA-EP0 were rescued by plaque purification,and the rescued strain and parent strain JS-2012 were subjected to one-step growth curve experiment and empty plaque test to analyze their in vitro biological characteristics.The results showed that in terms of plaque size and number and replication kinetics,the recombinant virus PRV-HA-EP0 was basically the same as that of the parent virus,while the replication rate of the EP0-deficient virus PRV-DEP0 decreased,indicating that HA labeled EP0 did not affect viral replication,while EP0-deficient viruses reduce viral replication capacity.Secondly,eukaryotic expression recombinant plasmid p3Flag-N-EP0 was constructed and transfected into HEK293 T cells.The indirect immunofluorescence and time image analysis were used to detect the expression localization of EP0,and the results showed that EP0 was mainly distributed in the nucleus,and EP0 protein had accumulated in the nucleus 6 hours after transfection.Then,the recombinant plasmid p3Flag-N-EP0 was transfected into HEK293 T cells and the deletion strain PRV-DEP0 infected porcine kidney cells PK-15 cells,and the host proteins interacting with EP0 in EP0 overexpression or virus infection were screened by IP experiments combined with LC-MS/MS protein spectroscopy,respectively.The results showed that 167 host proteins could interact with EP0 in EP0-overexpressed cells,while 172 could interact with them under viral infection.The enrichment analysis of the above interaction proteins by GO and KEGG showed that the interaction proteins under EP0 overexpression were mainly related to various signaling pathways such as genetic information processing and cell metabolism,among which the number of proteins in ribosomal components was the largest.Protein interaction network analysis showed that most of the interaction proteins of EP0 had transcriptional regulation.In the case of PRV infection with EP0 deletion,the interaction protein is also mainly involved in genetic information processing and cell metabolism of multiple signaling pathways,and after further comparison and analysis of the interaction proteins screened by the two,10 common differential genes were screened,and GO and KEGG analysis showed that the common differential genes were mainly localized in the cytoplasm,mainly containing ubiquitin ligase inhibitor activity.This suggests that EP0 may achieve mass proliferation by regulating the host ubiquitination pathway,thereby evading the "surveillance" of the host immune system.Finally,according to the above bioinformatics analysis,host proteins TUFM,HNRNPC and EIF3 E were selected for further study.The eukaryotic expression plasmids pc DNA-HATUFM,pc DNA-HA-HNRNPC,pc DNA-HA-EIF3 E were constructed,and they were cotransfected with p3Flag-N-EP0 to HEK293 T cells,and the analysis of Co-IP assay showed that HNRNPC could bind to EP0.Subsequently,different doses of EP0 were overexpressed to detect the expression of endogenous HNRNPC protein.The results showed that EP0 dosecorrelated decreased the expression of the HNRNPC protein.In summary,the deletion virus strain PRV-DEP0 and the HA-labled recombinant virus strain PRV-HA-EP0 were constructed,host proteins interacting with EP0 were screened,potential interaction proteins TUFM,HNRNPC,EIF3 E were selected,and the interaction between EP0 and HNRNPC protein was identified.The results lay a foundation for further exploration of the mechanism of EP0 transcriptional regulation.