The Research On Molecular Mechanisms Of PCBP2 Promoting The Replication Of FMDV

PCBP2(poly(rC)binding protein 2)belongs to a class of proteins that bind to poly(C)stretches of both RNA and DNA.Its roles are in maintaining mRNA stability and regulating translation.Meanwhile PCBP2 is a negative regulator in VISA-mediated antiviral signaling via E3 ubiquitin ligase AIP4 causing ubiquitin degradation of VISA to reduce production of IFN-β.Previous study have shown that PCBP2 can affect the replication of foot-and-mouth disease virus(FMDV),but the specific mechanism is unclear.Therefore,it is important to explore the mechanism that PCBP2 can affect the replication of foot-and-mouth disease virus for the prevention and control of FMD.1..HEK293 T cells were transfected with the FMDV plasmids VP2,2B,3A,L,VP0,2C,3C,3D and HA-PCBP2 plasmid.FMDV protein interacting with PCBP2 were detected by Immunoprecipitation and GST pull-down.The results showed FMDV structural protein VP0,2B,VP2,2C and 3D can interact with PCBP2.2.HEK293 T cell were transfected with reporter plasmid(IFN-β-Luc/ISRE-Luc),pRL-TK and PCBP2 and assessed as luciferase activity.The results showed PCBP2 is a negative regulator in SeV-mediated antiviral signaling and VISA-mediated antiviral signaling,and meanwhile showed a dose-dependent effect.HEK293 T cell were transfected with reporter plasmid,pRL-TK,HA-VISA FMDV plasmids L、VP2、2C、3D、VP0、2B and Myc-PCBP2 for 24 h,assessed as luciferase activity.The results showed 2C,3D,VP0 and 2B can promote the negative regulation of PCBP2 in VISA-mediated antiviral signaling via the Dual-Luciferase Reporter Assay.3.Immunoblot analysis of HEK293 T cells transfected with plasmids encoding Myc-tagged PCBP2 and various FMDV plasmids(2C,3D,VP0 and 2B)for 24 h.The results showed only FMDV VP0 increases specific degradation of VISA mediated by PCBP2.Further study found PCBP2 degraded VISA via proteasome pathway,VP0 degraded VISA via apoptotic pathway,PCBP2 and VP0 degraded VISA via apoptotic pathway;VP0 can increase the complex formation about PCBP2 and VISA,but VP0 can not increase the complex formation about PCBP2 and AIP4.FMDV mRNA level was higher after FMDV infection in VISA-deficient MEF cells comparing with wild-type MEF cells via Q-PCR Assay.The study first confirmed FMDV VP0 can increase degradation of VISA mediated by PCBP2 via VP0-PCBP2 interaction to reduce the production of IFN-β,and enhance the FMDV activity in cell...