感染腸病毒或表現腸病毒的 2B 蛋白會對細胞內鈣離子平衡造成劇烈變化。這包括減少內質網的鈣離子儲存量及活化細胞外鈣離子流入細胞質，最終使細胞質鈣離子濃度提升。細胞質鈣離子提升對病毒的複製格外重要，但鈣離子流入細胞質與病毒的複製機制的關係目前了解的很少。腸病毒 71 型的 2B 蛋白是一個鑲嵌在內質網的病毒穿孔蛋白質，它能消耗內質網的鈣離子並提升細胞質鈣離子濃度。本文中我們假設感染腸病毒 71 型或表現其 2B 蛋白能藉由消耗內質網鈣離子濃度來活化內質網鈣離子的感應蛋白(STIM1)，使 STIM1 蛋白與細胞膜上的ORAI1 蛋白結合以活化 ORAI1 通道，促使細胞外鈣離子流入細胞內。我們實驗證明感染腸病毒 71 型及表現其 2B 蛋白所誘導的細胞質鈣離子提升會被細胞內及細胞外鈣離子螯合劑所抑制。降低 STIM1 與 ORAI1 mRNA 的表現能降低細胞質鈣離子濃度並明顯抑制腸病毒 71 型複製晚期的蛋白與上清液的病毒效價，表示STIM1/ORAI1 在腸病毒 71 型的生命週期扮演著重要角色。藥物抑制 STIM1與 ORAI1 結合減少腸病毒 71 型所誘導的細胞質鈣離子提升和上清液的病毒效價。此外，我們建立並使用一個穩定表現 GFP-STIM1 蛋白的細胞株，去評估感染腸病毒 71 型後是否誘導 STIM1 活化(形成 puncta)。我們發現被腸病毒 71 型感染的細胞當中，位於內質網中的 STIM1 蛋白會持續活化並與細胞膜上的 ORAI1通道共位化。這些結果證實腸病毒 71 型誘導細胞質鈣離子提升及晚期的複製作用至少部分是靠 STIM1-ORAI1 的機制達成。此外，STIM1 和 ORAI1 可當作一個以宿主為導向的廣效型治療的靶的，用於治療腸病毒及與腸病毒複製機制類似的其他病毒。

Enterovirus (EV) infection or its 2B protein expression induces dramatic changes in cellular calcium homeostasis. These include decreased endothelium reticulum (ER) Ca2+ stores and activation of extracellular Ca2+ influx, ultimately causing an elevation in cytoplasmic Ca2+. Elevated cytoplasmic Ca2+ is absolutely required for virus replication, but the underlying mechanisms responsible for Ca2+ influx remain poorly understood. EV71 2B is an ER-localized viroporin, whose activity depletes ER calcium, which leads to Ca2+ influx. Herein, we exposed the possibility that EV71 infection or its 2B expression causes depletion of ER calcium through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1), subsequently activate Orai1, a plasma membrane Ca2+ channel that opens for extracellular Ca2+ entry. It was supported by our finding that EV71 infection- or viral 2B expression-induced cytoplasmic Ca2+ increase was reduced significantly by intracellular or extracellular Ca2+ chelator. Knockdown of STIM1 or Orai1 significantly reduced the virus-induced cytoplasmic Ca2+ as well as EV71 protein and viral titers at late stage of virus replication, indicating STIM1-Orai1 plays a critical role in virus replication. Treatments with pharmacological inhibitors of STIM1-Orai1 interaction also reduced the virus-induced Ca2+ increase and the viral titers in the supernatants. In addition, we established and used a stable green fluorescent protein-expressing STIM1 cell line as a biosensor to assess STIM1 activation (puncta formation) by EV71 infections. We found that STIM1 otherwise is localized in ER is constitutively active and colocalize with the Orai1 at plasma membrane in EV71infected cells. These data demonstrate that EV71- induced Ca2+ influx is at least partly mediated by STIM1-Orai1 machinery, which is required for the late stage of EV71 replication. Moreover, STIM1 and Orai1 may serve as novel targets for broad-spectrum host-directed therapeutics against infections of EVs as a group, and potentially other viruses that replicate via similar mechanisms.

摘要 1
Abstract 2
目錄 3
第一章 緒論 5
第一節 腸病毒的介紹 5
第二節 腸病毒71型 9
第三節 病毒與鈣離子的關係 10
第四節 Viroporin與腸病毒2B蛋白 11
第五節 三磷酸肌醇受體(IP3 receptor, IP3R)通道 12
第六節 鈣池調控的鈣離子通道 (Store-operated Ca2+ entry, SOCE) 的介紹、作用機制與病毒的關係 13
第七節 研究動機與方向 16
第二章 材料與方法 17
第一節 實驗材料 17
第二節 實驗方法 26
第三章 實驗結果 40
第一節 腸病毒71型感染細胞後其細胞質鈣離子隨時間變化 40
第二節 腸病毒71型的2B蛋白能使細胞質鈣離子上升，且提升的鈣離子可被細胞內鈣離子螯合劑 (BAPTA-AM) 及細胞外鈣離子螯合劑 (EGTA) 所抑制 41
第三節 降低STIM1、ORAI1 mRNA表現量能阻斷EV71誘導的細胞質鈣離子濃度上升 42
第四節 降低STIM1、ORAI1 mRNA表現量能在第12小時抑制腸病毒71型的基因體複製 43
第五節 降低STIM1、ORAI1 mRNA表現量能抑制腸病毒71型的釋放作用 44
第六節 使用ORAI1通道抑制劑SKF96365 、Synta66及AnCoA4能阻斷腸病毒71型誘導細胞質鈣離子提升 45
第七節 使用ORAI1通道抑制劑SKF96365及AnCoA4能在第6小時抑制腸病毒71型的基因體複製，Synta66則對腸病毒71型的基因體複製沒有抑制效果。 46
第八節 使用ORAI1通道抑制劑SKF96365 、Synta66及AnCoA4能明顯抑制腸病毒71型的釋放作用 47
第九節 感染腸病毒71型會造成STIM1蛋白進行聚集化作用形成puncta 48
第十節 感染腸病毒71型會造成STIM1蛋白與ORAI1蛋白結合以活化ORAI1通道 49
第四章 討論 50
第五章 圖表 53
第六章 參考文獻 63
第七章 附錄 69

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