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目的:比较并探讨日本脑炎病毒Beijing-1株和登革2型病毒43株对宿主细胞内IFN-α介导的信号转导通路抑制作用的机制。方法:采用含萤火虫荧光素酶(Luciferase,Luc)报告基因的重组载体pISRE-Luc,通过检测IFN刺激应答元件(IFN-stimulated response element,ISRE)活性对病毒感染细胞内IFN-α介导的JAK-STAT信号转导通路的抑制作用进行定量分析。利用间接免疫荧光法观察在IFN-α作用下病毒感染细胞内STAT1分子的分布情况。进一步采用Western印迹分别检测在这两种病毒感染状态下宿主细胞内STAT1、JAK1和TYK2的磷酸化水平。结果:感染日本脑炎病毒和登革病毒的细胞在IFN-α作用下,ISRE活性与对照组相比均显著下降,而且日本脑炎病毒对宿主细胞内ISRE活性的抑制程度明显强于登革病毒;进一步研究发现,日本脑炎病毒可通过抑制JAK1和TYK2两种激酶的活性,降低STAT1的磷酸化水平,阻碍STAT1的核转运;而登革病毒则只抑制TYK2激酶的活化,降低STAT1的磷酸化及核转运水平。结论:日本脑炎病毒和登革病毒可通过不同的作用机制抑制IFN-α介导的JAK-STAT信号转导通路。
OBJECTIVE: To compare and investigate the mechanism of inhibition of IFN-α-mediated signal transduction pathway by host Japanese encephalitis virus strain Beijing-1 and dengue virus type 2 in host cells. Methods: The recombinant plasmid pISRE-Luc containing firefly luciferase (Luc) reporter gene was used to detect IFN-α-mediated JAK activity by detecting IFN-stimulated response element (ISRE) -STAT signal transduction pathway inhibition quantitative analysis. Indirect immunofluorescence was used to observe the distribution of STAT1 in virus-infected cells under the action of IFN-α. Further Western blotting was used to detect the phosphorylation level of STAT1, JAK1 and TYK2 in host cells under the infection of these two viruses respectively. Results: The activity of ISRE in IFN-α-infected Japanese encephalitis virus and dengue virus cells were significantly decreased compared with the control group, and the Japanese encephalitis virus inhibited the expression of ISRE activity more significantly in host cells Further studies showed that Japanese encephalitis virus could inhibit the phosphorylation of STAT1 and block the nuclear translocation of STAT1 by inhibiting the activity of both JAK1 and TYK2. However, dengue virus only inhibited the activation of TYK2 and decreased the expression of STAT1 Phosphorylation and nuclear transport levels. Conclusion: Japanese encephalitis virus and dengue virus can inhibit IFN-α-mediated JAK-STAT signal transduction pathway through different mechanisms of action.