口蹄疫病毒结构蛋白VP0抑制I型干扰素信号通路的激活

Structural protein VP0 of foot-and-mouth disease virus inhibits type I interferon signaling pathway

摘要:

[目的]研究口蹄疫病毒（FMDV）结构蛋白VP0对I型干扰素信号通路的影响。[方法]通过反转录PCR构建VP0真核表达载体，利用Western blotting验证VP0蛋白转染HEK-293T细胞后的表达情况；Real-time PCR检测VP0蛋白对FMDV在BHK细胞上复制的影响，检测VP0蛋白对SeV诱导的干扰素信号通路分子RIG-I、IRF3、IFN-β及下游刺激基因ISG15、ISG20表达的影响；双荧光素酶报告基因检测系统检测VP0蛋白对SeV诱导的IFN-β和NF-κB启动子激活以及对RIG-I样受体（RIG-I-like receptors，RLRs）信号通路分子激活IFN启动子的影响；免疫共沉淀检测VP0蛋白与RLRs信号通路中关键分子的相互作用。[结果]成功构建了pCAGGs-VP0真核表达载体，可以在HEK-293T细胞中表达；FMDV感染后的4-6 h，VP0蛋白显著促进FMDV在BHK细胞上的复制（P<0.01或P<0.05）；VP0蛋白明显抑制干扰素下游刺激基因的表达（P<0.01或P<0.05）。在双荧光素酶报告基因检测实验中，VP0蛋白抑制SeV诱导IFN-β和NF-κB的活化具有剂量依赖性（P<0.01），并对RIG-I、MDA5、VISA、TBK1和IRF3介导的IFN-β产生具有抑制作用，但是对IRF7没有明显的影响。免疫共沉淀显示VP0蛋白可与IRF3发生相互作用。[结论]证实VP0蛋白可以通过与IRF3相互作用来抑制I型干扰素信号通路的激活。

关键词:口蹄疫病毒;VP0蛋白;I型干扰素;IRF3

Abstract:

[Objective] To explore the effects of foot-and-mouth disease virus (FMDV) structural protein VP0 in type I IFN signaling.[Methods] A recombinant VP0 protein was constructed and expressed in eukaryotic cells followed by FMDV infection. The influence of VP0 protein during FMDV replication in BHK cells was determined by RT-PCR and the mRNA levels of IFN-β, RIG-I, ISG15, ISG20 and IRF3 was assessed. The dose-dependent effects of VP0 protein on Sendai virus (SeV)-triggered activation of IFN-β and NF-κB promoter as well as on RLRs-mediated activation of IFN-β promoter were examined by luciferase assay. Co-immunoprecipitation was done to detect the interaction of VP0 protein and the components of the RLRs signaling pathway.[Results] The recombinant VP0 protein was successfully expressed in HEK-293T cells and confirmed by Western blotting. At 4-6 h post-infection, VP0 protein significantly promoted the replication of FMDV in BHK cells (P<0.01 or P<0.05). VP0 protein clearly inhibited the gene expression level of IFN-β, RIG-I, ISG15, ISG20 and IRF3 (P<0.01 or P<0.05), respectively. In luciferase assays, FMDV VP0 protein distinctly inhibited SeV-triggered activation of the IFN-β and NF-κB promoters in a dose-dependent manner. The interferon regulatory factor 3 (IRF3)-activated IFN-β promoter and its upstream components, RIG-I, MDA5, VISA and TBK1 were also down-regulated in the presence of VP0 protein. The inhibition of IFN-β promoter induced by IRF7 was not observed. Furthermore, Co-immunoprecipitation showed that VP0 interacted with IRF3 protein in HEK-293T cells.[Conclusion] Our results indicated that VP0 may inhibit the activation of type I IFN signaling pathway via interaction with IRF3.

Key words:foot-and-mouth disease virus;VP0 protein;IFN-β;IRF3