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

【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p CAGGs-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。

[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-13 and NF-κB promoter as well as on RLRs-mediated activation of IFN-13 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.