Esterase D Enhances Type â…  Interferon Signal Transduction To Suppress Foot-and-mouth Disease Virus Replication

During FMDV infection, the sensing of viral RNAs are mainly recognized by MDA5 which is one of the pattern-recognition receptors(PRRs). VISA(virus-induced signaling adaptor) is recruited to MDA5 to mediate signal transduction. VISA interacts with TRAF6 to activate the IKK complex, which leads to phosphorylation and degradation of IkBα, thereby releasing NF-?B into nucleus. VISA also recruits MITA and promotes phosphorylation of IRF3 by TBK1. The phosphorylated IRF3 forms dimer and transfers into nucleus. The activated NF-?B and IRF3 trigger the transcription and translation of type I IFNs and various antiviral interferon-stimulated genes(ISGs), which promotes innate antiviral immune responses.The enzymatic activities of esterase D(ESD) are involved in many human diseases. However,whether ESD regulates the innate immune signal transduction and plays a role in cellular antiviral response has not been described to date. Our previous transcriptome analysis revealed that ESD mRNA expression was significantly up-regulated in FMDV-infected cells compared with mock-infected cells,suggesting a potential role of ESD in FMDV infection. In this study, we investigated the state of ESD after FMDV infection, and the results indicated that ESD transcripts and protein abundance were significantly up-regulated after FMDV infection. Then we amplified swine ESD gene and constructed an eukaryotic expression plasmid containing the swine ESD coding sequence fragment and performed an overexpression assay, which indicated that FMDV replication was significantly suppressed in ESD up-regulated cells, and this inhibitory effect was dose-dependent. To confirm the antiviral role of ESD against FMDV, we further used RNAi to down-regulate ESD expression in PK-15 cells, and the results indicated that FMDV replication was significantly enhanced in ESD knockdown cells. According to the overexpression and knockdown assays, we demonstrated that ESD inhibits virus replication during FMDV infection.To explore the mechanism of ESD-mediated antiviral role, the mRNA expression levels of several antiviral IFN-stimulated genes(ISGs) were detected by qPCR. The ISGs, including ISG15, MX1,ISG54, RIG-I, PKR and GBP1, were significantly up-regulated in ESD overexpression cells compared with vector-transfected cells after FMDV infection; and knockdown of ESD also decreased the expression of these ISGs during FMDV infection. To explore whether ESD regulates type I IFN signaling, IFN-β promoter luciferase assay was carried out, and the results showed that overexpression of ESD protein significantly increased the Se V-induced promoter activities of IFN-β. To further explore the mechanism of the ESD-enhanced type I IFN signaling, the type I IFN pathway adaptor molecules mediated IFN-β promoter luciferase assays were performed, and the results showed that overexpression of all the adaptor molecules of the RLR signaling pathway significantly activated the promoter of IFN-β.Activation of the IFN-β promoter induced by IRF3 or its upstream molecules(RIG-I-CARD, MDA5,VISA, and TBK1) was enhanced by ESD protein. In contrast, activation of the IFN-β promoter induced by IRF7(downstream of TBK1) was not affected by ESD protein. It implied that IRF3 was a target of ESD to enhance IFN-β signal transduction. Therefore, the expression of MDA5, VISA, TBK1,phospho-TBK1, IRF3, phospho-IRF3 was detected by western blotting after FMDV infection, and the results showed that expression of VISA, TBK1 and IRF3 was not affected by ESD protein, and phosphorylation of IRF3 was significantly increased by upregulation of ESD. We also detected the expression and phosphorylation levels of these adaptor molecules in ESD knockdown cells by western blotting, and the results also indicated that ESD was essential for FMDV-triggered IRF3 phosphorylation and suppression of viral replication. These results demonstrate that ESD enhances IFN-β signal transduction and suppresses viral replication through promoting phosphorylation of IRF3.In summary, we report here that ESD has an antiviral role against FMDV infection. In addition, we describe a novel mechanism by which ESD enhances the phosphorylation of IRF3 to regulate the type I IFN signaling pathway and promote the expression of antiviral ISGs, such as ISG15, PKR, MX1, ISG54,RIG-I and GBP1. These findings indicate that ESD plays an important role in enhancement of type I IFN-mediated immune response against FMDV infection.