臺灣博碩士論文加值系統

腸病毒 （EV）由脊髓灰質炎病毒 (poliovirus)，柯薩奇病毒 (Coxsackievirus)，伊科病毒 (Echovirus)和鼻病毒 (Rhinovirus)屬於小核糖核酸病毒科的物種組成。其中，EV71被認為是與輕微的手足口病和幼兒嚴重神經系統表現相關的最致病的病原體之一。宿主細胞鈣 （Ca2+）信號越來越被認為在包括腸道病毒在內的一系列病毒的病毒複製中起重要作用。事實上，一些EV物種的研究表明，EV感染導致細胞內Ca2 +升高是病毒複製所必需的。而在EV71感染隨後的Ca2+升高與鈣蛋白酶-1和-2的活化有關，它們都屬於哺乳動物中普遍表達的Ca2 +依賴性非溶酶體半胱氨酸蛋白酶家族。研究表明，鈣蛋白酶參與柯薩奇病毒B3和伊科病毒1型複製中的病毒基因組複製和病毒蛋白質合成，並且鈣蛋白酶在病毒複製的晚期介導EV71所誘導的細胞凋亡。然而，鈣蛋白酶是否參與EV71複製的早期階段並且其潛在機制尚不清楚。因此，在本研究中，我們研究了鈣蛋白酶在EV71複製週期中的作用機制。結果顯示，在EV71感染期間，當處理鈣蛋白酶-1 / -2抑製劑MDL28170或鈣蛋白酶1/2 shRNA沉默時，病毒蛋白和病毒基因組以劑量依賴性方式降低。通過抑製劑或基因沉默，II-血影蛋白裂解反映著鈣蛋白酶活性的減少，表明鈣蛋白酶確實介導EV71的複制。為了研究鈣蛋白酶與EV71複製有關的潛在途徑，我們使用生物信息軟件來希望解決這個問題。在預測的目標中，細胞外調節的激酶ERK 1/2最有可能出現，因為它們與EV71感染細胞的促進存活狀態有關。由此，我們發現透過鈣蛋白酶抑製劑MDL28170或鈣蛋白酶-1 / -2基因沉默可導致EV71感染引起的ERK 1/2磷酸化降低。總之，我們發現了一種可能與鈣蛋白酶介導的EV71複製相關的新機制。 該結果有助於理解鈣蛋白酶介導的EV71複製的分子機制，並且可以作為開發具有相似複製機制的腸道病毒71型和其他病毒的廣效型治療的基礎。

Enteroviruses (EVs) consist of poliovirus, coxsackievirus, echovirus, and rhinovirus species that belong to the family Picornaviridae. Among them, EV71 is considered as one of the most virulent pathogens associated with mild hand, foot, and mouth disease and severe neurological manifestations in young children. Host cell calcium (Ca2+) signals are increasingly recognized to play important roles in viral replication for a range of viruses including enteroviruses. Indeed, studies from some EV species showed that EV infections resulted in an elevation in intracellular Ca2+ that is absolutely required for virus replication. The consequent Ca2+ elevation upon an EV71 infection is associated with activation of calpain-1 and -2, both belong to the family of Ca2+-dependent, non-lysosomal cysteine proteases expressed ubiquitously in mammals. Studies have shown that calpains are involved in the viral genome replication and viral protein synthesis in coxsackievirus B3 and echovirus 1 replication, and that calpains mediate EV71-induced apoptosis in the late stage of viral replication. However, whether calpains are involved in the earlier stage of EV71 replication and the underlying mechanism is not well understood. So in this study we have explored the mechanism of calpain in the replication cycle of EV71. The results showed that the viral protein and viral genome decreased in a dose-dependent manner upon the treatment of a calpain-1/-2 inhibitor MDL28170 or calpain 1/2 shRNA silencing during EV71 infections. Calpain activities reflected by the cleavage of αII-spectrin were reduced by the inhibitor or the knockdown, indicating that calpains are indeed to mediate the replication of EV71. To investigate the potential pathway that calpains are implicated in EV71 replication, we used the bioinformatic analysis to hopefully resolve the question. Among the predicted targets, extracellular regulated kinases ERK 1/2 emerged as the most likely ones as they have been implicated in the pro-survival state of EV71-infected cells. Indeed, we further showed that the virus-induced ERK1/2 phosphorylation was reduced significantly by calpain inhibitor MDL28170 or calpain-1/-2 knockdown, indicating that calpains may up-regulate EV71 replication through ERK1/2 phosphorylation. Taken together, we have uncovered a novel mechanism that may correlate calpain-mediated EV71 replication. This result contributed to understanding of molecular mechanism underlying calpain-mediated EV71 replication, and may serve as a basis for development of the broad-spectrum treatment of enterovirus 71 and other viruses with similar replication mechanism.

摘要 ii
Abstract iii
目錄 iv
第一章　緒論 1
第一節　腸病毒概論 1
第二節　腸病毒71型的臨床病徵和流行病學 1
第三節　腸病毒的基因體結構 2
第四節　腸病毒的複製週期 3
第五節　病毒對於鈣離子濃度的調控 4
第六節　鈣蛋白酶與腸病毒造成細胞死亡的關係 5
第七節 鈣蛋白酶與腸病毒的關係 7
第八節　研究緣由與目的 8
第二章 材料與方法 9
第一節 實驗材料 9
壹、細胞與病毒 (Cell line and Virus) 9
貳、試劑、藥品與溶液 (Reagent, Drugs and Solution) 9
參、試劑套組與酵素 (Kits and Enzymes) 12
肆、引子 (primer) 13
伍、質體 (Plasmids) 14
陸、抗體 (Antibodies) 14
第二節 實驗方法 15
一、細胞株的培養與操作 15
二、病毒的培養與定量 16
三、細胞存活率試驗 (MTS Cell Proliferation Colorimetric Assay) 17
四、質體DNA轉染 18
五、Lentivirus 濃縮 18
六、shRNA stable knockdown 18
七、即時定量聚合酶連鎖反應 (Real-time PCR, qPCR) 20
八、西方墨點法 (Western blot) 22
第三章 實驗結果 25
第一節 鈣蛋白酶的抑制劑可以抑制腸病毒71型的病毒蛋白 25
第二節 剔除鈣蛋白酶會抑制腸病毒71型的複製 26
第三節 使用Search Tool for Interactions of Chemicals (STITCH)預測鈣蛋白酶1和2跟腸病毒71型複製的相關蛋白 27
第四節 鈣蛋白酶抑制劑能降低ERK1/2磷酸化且有劑量依頼性的效果 28
第五節 鈣蛋白酶參與腸病毒71型所誘導ERK1/2的磷酸化路徑 29
第四章 討論 30
第五章 圖表 34
第六章 參考文獻 43
第七章 附錄 49
附錄一、腸病毒屬之分類[1] 49
附錄二、腸病毒之結構[18] 50
附錄三、腸病毒之基因體[20] 50
附錄四、腸病毒71型在感染期間提升細胞內鈣離子濃度 51
附錄五、使用細胞內鈣離子螯合劑抑制腸病毒71型的複製 51
附錄六、鈣蛋白酶結構[40] 52
附錄七、鈣池調控鈣離子流入之機制[47] 52
附錄八、鈣蛋白酶活性下降抑制腸病毒71型病毒複製之路徑 53

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