Effects Of Host ARFGAP1 On CSFV Replication And Interaction With NS5A

Classical Swine fever(CSF)caused by the Classical swine fever virus(CSFV)is a multi-system,highly contagious and economically important disease that causes significant economic losses worldwide.Exploring cost-effective prevention methods depends largely on a clear understanding of the pathogenesis of CSFV.Studies found that Coat protein complex?(COP ?)can participate in the regulation of virus replication and COP ? is closely related to CSFV replication.ADP ribosylation factor GTPase-activating protein 1(ARFGAP1)is a key regulatory protein of COP I.ARFGAP1 participates in the formation of COP ? and clathrin-coated vesicles.ARFGAP1 also plays a role in viral replication.CSFV NS5A is an unstructured protein closely related to viral replication,but it was not known whether ARFGAP1 interacts with NS5A.Therefore,the purpose of this study was to investigate the effect of ARF GAP 1 protein on CSFV replication and its interaction with NS5A,and the following results were obtained.(1)ARFGAP1 positively regulates CSFV replication.Treatment of cells with different concentrations of ARF GAP1 inhibitor(QS11)or treatment of cells with the same concentration of QS11 at different time points significantly inhibited CSFV replication and progeny virus production(P<0.05,P<0.01);RT-qPCR and IFA detection found that overexpression of ARFGAP1 significantly promoted CSFV replication and progeny virus production,while interference with ARFGAP1 significantly inhibited the CSFV replication and progeny virus production(P<0.05,P<0.01).Our findings revealed that ARFGAP1 can positively regulate CSFV replication.(2)CSFV infection promotes the mRNA and protein expression of ARFGAP1.The mRNA and protein expression of ARFGAP1 in CSFV infected cells were detected by RT-qPCR and Western blot respectively.The results showed that CSFV infection increased the mRNA and protein expression of ARFGAP1.(3)ARF GAP1 protein interacts with CSFV NS5 A protein.Co-Immunoprecipitation(Co-IP)assay showed the interaction between ARFGAP1 and CSFV NS5A in cells.while GST pull-down found the direct interaction between ARF GAP1 and CSFV NS5A protein.The colocalization of ARF GAP1 and NS5A protein was observed by Confocal laser scanning microscope(CLSM).In summary,studies found that ARFGAP1 protein in host cells promotes the replication of CSFV and interacts with CSFV NS5A.The results provide new scientific data for understanding the effect of ARFGAP1 on CSFV replication and its interaction with CSFV NS5A.