Foot-and-mouth Disease Virus Protein 3A Interacts With Annexin-1 To Regulate Type Ⅰ Interferon Signal Pathway

Foot-and-Mouth Disease Virus(FMDV)is a single-strandedpositive-sense RNA virus that causes Foot-and-Mouth Disease(FMD)in cattle,pigs,and various clovenhoofed animals.Viral infections are sensed by pattern-recognition receptors(PRRs)of the innate immune system that recognize pathogen associatedmolecular patterns(PAMPs).RNAs from FMDV as PAMPsare recognized bycytosolic RIG-I-like receptor(RLR)family members RIG-I(retinoic acid-inducible gene I)andMDA5(melanoma differentiation-associated gene-5).Recognitionof viralRNAs by thePRRs triggers a series of signaling eventsthat lead to induction of typeⅠinterferons(IFNs).TypeⅠIFNs activate the JAK(Janus kinase)-STAT(signal transducer and activator of transcription)signal transduction pathways,leading to transcriptional induction of a wide range of downstream antiviralgenes and subsequent innate antiviral response.The recombinant Myc-ANXA1-pCAGGs was successfully constructed.HEK293 T,Hela,BHK21andPK15 cells were transfected withthe Myc-ANXA1-pCAGGsrecombinant plasmid,viral protein plasmid andplasmidofthe type Ⅰ IFNs pathway adaptor molecules.Technology method ofQPCR,Confocal microscopy,Immunoprecipitation,Western blottingand Luciferase assays was used to investigation of FMDV protein 3A interacts with Annexin-1(ANXA1)and the regulatory effect on IFNs production.1.ANXA1 was cleavaged during FMDV infection PK15 cells.Expression of ANXA1 protein abundance were significantly up-regulated after SeV infection HEK-293 T and Hela cells,showing ANXA1 participated in the antiviral response of cells.2.Overexpression of ANXA1 significantly suppressed FMDV replication and knockdown of ANXA1 expression enhanced virus replication.3.Overexpression of ANXA1 protein significantly increased the activation of typeⅠIFNs pathway and enhanced the SeV-induced expression of IFNsin a dosedependent manner.FMDVprotein 3Asignificantly inhibited ANXA1-induced expression of IFNs during SeV infection.4.Activation of the IFN-β promoter induced by RIG-I,MDA5,TBK1,IRF3 and IRF7 was enhanced by ANXA1 protein.In contrast,activation of the IFN-β promoter induced by VISA(upstream of TBK1)was not affected by ANXA1 protein.To further explorephosphorylation of TBK1 and IRF3 were significantly increased by upregulation of ANXA1,and phosphorylation of TBK1 and IRF3 were significantly decreased by knockout of ANXA1 gene.This result demonstrated that ANXA1 enhanced typeⅠsignal transduction through promoting phosphorylation of TBK1 and IRF3.5.The regulatory effect of ANXA1 onexpression levels of several antiviral IFNstimulated genes(ISGs)oftype Ⅰ IFNs pathwaydownstream.The ISGs,including RNATES,CXCL10,ISG54 and ISG56,were significantly up-regulated in ANXA1 overexpression cells.The study first demonstrate that ANXA1 suppresses FMDV replication through enhancingFMDV-triggered phosphorylation of TBK1 and IRF3 to regulate the typeⅠIFNs signaling pathway and promotes the expression of antiviral ISGs.