PCBP2 Interacts With ISG15 And The Preliminary Exploration Of Its Effect On Replication Of Classical Swine Fever Virus

Classical swine fever(CSF),also known as Hog Cholera(HC),is one of the most important infectious diseases of swine and wild boar caused by classical swine fever virus(CSFV).CSF is listed as class I infectious disease in China,and listed as a national mandatory immunization program.CSFV is one of the members of the Pestivirus genus of the Flaviviridae family,which is a single-stranded positive RNA virus with an envelope.CSFV infection activated multiple signal pathways and induced transcriptional expression of related cytokines in PK-15 cells,but the expression of IFN-α/β was suppressed.Interestingly,CSFV inhibited the expression of IFN-α/β and its downstream proteins in PK-15 cells,but upregulated the expression of ISG15,which indicating that the expression of ISG15 might be regulated by other signaling pathways.However,it is still unclear how ISG15 exerts its resistance against CSFV.In the previous study of the mechanism of ubiquitin-like protein ISG15 against CSFV in our laboratory,more than 100 proteins which potentially interacted with ISG15 were obtained by immunoprecipitation and mass spectrometry.In this study,polycytosine binding protein 2(PCBP2)was selected as the research protein according to the software analysis and screening.Previous studies in our laboratory showed that the CSFV genome RNA(gRNA)activated the expression of RIG-1,MAVS,IRF1 and ISG15 by a dose-dependent manner.Some studies reported that PCBP2 protein could bind with ubiquitin to degrade MAVS.ISG15 is a kind of ubiquitin like protein,our research aim to explore whether ISG15 binds to PCBP2 through a similar function,and thereby plays an antiviral role.In this experiment,the eukaryotic expression vectors of ISG15 and PCBP2 were successfully constructed,and the expressions of the recombinant proteins were detected by western blot.The results of the CO-IP experiment proved that ISG15 interacted with PCBP2 in vivo.Simultaneously the prokaryotic expression vectors ISG15 and PCBP2 were successfully constructed,and the conditions for inducing the expression of GST-ISG15 and GST-PCBP2 fusion proteins were explored.The result of GST-Pulldown experiment demonstrated that PCBP2 interacted with ISG15,in vitro.The fluorescence images clearly pointed out that PCBP2 protein and ISG15 protein existed at the same site in co-transfected PK-15 cells.In order to further study the effect of the interaction between PCBP2 and ISG15 on the re plication of CSFV.The experiment showed that the change of PCBP2 was correlated with ISG15 to some extent without virus stimulation.Compared with the control group,the replication of CSFV was enhanced after knockdown of PCBP2 in PK-15 cells.Some studies proved that PCBP2 could regulate the RNA replication of some viruses.PCBP2 may bind to CSFV gRNA by interacting ISG15.The transcription level of ISG15 is not affected while the PCBP2 gene is disturbed,but its antiviral effect need to be executed through other routes.In summary,this study verified that ISG15 could directly interact with PCBP2,and discovered that the PCBP2 was negatively correlated to the replication of CSFV.These date will be helped to further research on the mechanism of pathogenic and innate immune of CSFV.