Studies On Molecular Mechanism That Nonstructural Protein 4 Of PRRSV Cleaves STAT2

Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by the Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).And it is a viral disease that causes enormous losses in the world’s pig production.The study found that PRRSV can evade host immunity by encoding proteins to suppressing natural immunity.In this study,we found that nsp4 encoded by PRRSV can cleave the key signal molecule STAT2 of JAK-STAT signaling pathway.Further analysis of the details of nsp4 cleave STAT2,and explored its role in PRRSV regulation of natural immune response.The main contents are as follows: 1.PRRSV inhibited IFN-α-induced ISGs expressionQuantitative real-time PCR analysis showed that m RNA level of 2’,5’-OAS,ISG15,ISG56 and ISG60 which induced by IFN-α was significantly inhibited by PRRSV infection PAMs or MARC-145 cells.Dual luciferase reporter assay showed that ISRE-Luc activation was significantly inhibited by PRRSV infection with MARC-145 cells.In order to screen for protein that inhibits ISRE activity,the eukaryotic expression plasmids of all structural and non-structural proteins encoded by PRRSV were co-transfected with the luciferase reporter plasmids ISRE-Luc and p GL-4.74 to HEK-293 T cells.Dual luciferase reporter assay showed that the non-structural proteins nsp1 a,nsp1b,nsp4,nsp11,nsp12 and structural proteins GP3 and N of PRRSV could inhibit the activity of IFN-α-induced ISRE promoter,and the Inhibition level of nsp4 was obviously.2.The serine protease catalytic activity is essential for nsp4 to inhibit ISRE activationThe eukaryotic expression plasmid nsp4 and luciferase reporter plasmid ISRE-Luc were co-transfected with HEK-293 T or 3D4 cells,the dual luciferase assay showed that nsp4 could significantly inhibit IFN-α-induced ISRE promoter activity in a dose-dependent manner.meanwhile,the double luciferase reporter system demonstrated that nsp4 inhibits IFN-α-induced ISRE promoter activity depending on its serine protease activity.3.PRRSV-encoding nsp4 protein cleaves STAT2 at E719In this study,we found that over-expression nsp4 could cleave the key protein STAT2 in JAK-STAT signaling pathway in a dose-dependent manner,but has no effect on m RNA level of STAT2 protein.After exposure to MARC-145 cells by PRRSV,endogenous STAT2 was found to be degraded by Western Blot,but no cleavage was detected.It was also proved that nsp4 cleavage of STAT2 did not depend on ubiquitin-proteasome pathways and apoptotic pathways,but rather dependent on its own serine protease activity.furthermore,the cleavage site was identified at E719.4.The PRRSV-encoded nsp4 protein antagonizes the JAK-STAT signaling pathwayWe discovered that,nsp4 eukaryotic over-expression plasmid was co-transfected with STAT1 and STAT2 eukaryotic over-expression plasmids.Co-IP showed that the interaction between STAT1 and STAT2 was significantly attenuated by PRRSV-encoding nsp4 protein.The full-length or truncated mutants of STAT2 were co-transfected with STAT1 and IRF9 for double luciferase assay,and the result showed ISRE was only activated by full-length instead of truncated mutants of STAT2 indicating that cleavage of STAT2 by nsp4 severely inhibited JAK-STAT signaling pathway.In conclusion,our study first reveals the important role of PRRSV-encoding nsp4 protein in antagonizing JAK-STAT signaling pathway.It is found that PRRSV-encoding nsp4 protein is dependent on its own serine protease activity to cleave STAT2 protein which is important signal molecules in JAK-STAT signaling pathway,and then prevent delivering to the downstream,inhibiting ISRE promoter activity and ISGs transcription expression.At the same time,we confirmed that nsp4 cleaved STAT2 at E719.This study deepens our understanding of the pathogenesis and immune escape mechanism of PRRSV,providing the theoretical basis for PRRSV vaccine research and development.