| 摘要: | 日本腦炎病毒屬於以蚊子為傳染媒介的黃質病毒科，它能造成人類與許多動物中樞神經的感染，甚至形成腦膜炎。流行在亞洲地區，每年病例約三萬至五萬人。目前在台灣地區由於疫苗的廣泛施行，從1998至2007日本腦炎通報病例數每年約3-4百人，確定病例每年約20-30例。在治療方面並沒有針對日本腦炎之抗病毒藥物，只能依病情給予支持性療法。日本腦炎病毒的非結構蛋白NS5在日本腦炎病毒基因組裡屬最大段也是具高度保守的序列，它與甲基轉移酶以及依附RNA型RNA聚合酶有關，是病毒複製時非常重要的蛋白。在過去的研究指出，日本腦炎病毒NS5蛋白能夠藉由阻斷Jak-Stat分子訊息傳遞的方式去抵抗第一型干擾素的作用。本研究論文之目的在確認NS5蛋白為干擾素的拮抗物，並利用二維電泳圖譜以及基質輔助雷射脫附游離-飛行時間質譜之蛋白質體學方法去比較NS5表現蛋白細胞經干擾素IFN-β處理作用後相關蛋白變化以了解NS5蛋白在細胞內的分子機制。首先利用流式細胞儀檢測Annexin V與PI染色後的細胞，發現NS5蛋白表現的細胞能夠抑制IFN-β所誘導之細胞凋亡。另一方面以細胞內冷光報導基因試驗顯示NS5蛋白具有抑制干擾素所引起的ISRE啟動子活性的作用。同時利用即時定量聚合酶連鎖反應試驗發現NS5蛋白對於IFN-β所誘發產生的抗病毒蛋白基因PRK及OAS有抑制表現的現象。接著經二維電泳及質譜儀分析發現與空載體對照組細胞相比，在NS5蛋白表現細胞加入干擾素誘發表現上升的蛋白有：Fascin、Peroxiredoxin-1及Cyclophilin A等5個蛋白；而表現下降的蛋白如：Heterogeneous nuclear ribonucleoprotein L 及Stress-induced- phosphoprotein 1等16個蛋白。同時我們利用西方墨點法確認Cyclophilin A在NS5蛋白表現細胞中含量較空載體對照細胞高。最後我們加入cyclophilin A抑制劑Cyclosporin A能夠回復被NS5蛋白所抑制的干擾素下游ISRE啟動子之活性，以及抑制ERK1/2的磷酸化。此外利用即時定量聚合酶連鎖反應與流式細胞儀發現Cyclosporin A具有對抗日本腦炎病毒複製以及抗病毒所誘發的細胞凋亡能力。藉由病毒蝕斑得知Cyclosporin A處理其他種類細胞株有同樣的抗日本腦炎病毒反應。本論文的發現或許能夠提供一個治療日本腦炎的新療法。
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, infects central nervous system in human and animal, resulting in encephalitis. Thirty to fifty thousand JE cases occur annually in Asia. A mass vaccination of Nakayama strain JE inactivated vaccine has been launched since 1968 in Taiwan, therefore only sporadic JE cases occur between August and October in Taiwan. Specific and effective anti-viral drugs for JE treatment are not available, thus JE treatment is supportive. JEV non-structural protein 5 (NS5), the largest and most conserved flavivirus protein, has methyltransferase and RNA-dependent RNA polymerase activities, being critical for virus replication. In addition, JEV NS5 protein has been demonstrated the inhibitory effect on the interferon (IFN)-α/β response by blocking JAK-STAT singaling pathway. The goal of this study is to investigate the molecular mechanism of the IFN antagonist function by JEV NS5 using two-dimensional electrophoresis (2-DE) and mass spectrometric (MS) identification, developing the novel targets for the JE treatment. Initially, the NS5-expressing cells, but not vector control cells were resistant to the effects of IFN-β treatment, such as apoptosis, the promoter activity of the interferon stimulation response element (ISRE), and the mRNA expression of oligoadenylate synthetases (OAS) and protein kinase R (PKR). Comparison of proteome profiling between NS5-expressing cells and vector control cells revealed that the expression of JEV NS5 protein resulted in the up-regulation of 5 identified proteins including fascin, Peroxiredoxin-1, and cyclophilin A, and down-regulation of 16 identified proteins including heterogeneous nuclear ribonucleoprotein L and stress induced phosphoprotein 1 in human medulloblastoma TE671 cells. Western blotting confirmed the up-regulation of cyclophilin A in NS5-expressing cells in absence or presence of the IFN-β treatment. Finally, cyclosporin A, a cyclophilin inhibitor restored IFN-β induced ISRE promoter activity and inhibited the phosphorylation of ERK1/2 in NS5-expressing cells in presence of the IFN-β treatment. Importantly, cyclosporin A inhibited the JEV replication and decreased JEV-induced apoptosis in TE671 cells and BHK-21 cells. This study demonstrate unique proteome profiling of NS5-expressing cells in absence and presence of the IFN-β treatment, identifying cyclophilin A as the therapeutic target against JEV. The results might provide a new therapeutic strategy for JEV infection. |
