臺灣博碩士論文加值系統

腸病毒71型(EV71)目前在亞太地區包含中國、台灣、新加坡、與馬來西亞等地為一項重要的公共健康問題。腸病毒71型是造成兒童手足口症中一項重要的致病原，嚴重時甚至會侵害中樞神經系統造成病童死亡。目前並沒有開發出有效的抗病毒藥物或疫苗，更多的研究必須進行以更深入了解病毒與宿主間的交互作用。在過去，對於病毒的研究主要著重在細胞內與宿主蛋白質之間的關係，然而，較少研究針對宿主細胞外，與病毒一同釋放之宿主蛋白質，在病毒生活史中的重要性。本研究中，藉由液相層析/質譜分析鑑定出幾個細胞受病毒感染所分泌之宿主蛋白，其中包含了HSP90β，但並不包含HSP90α。藉由蔗糖梯度離心，免疫共沉澱法，電子顯微鏡觀察發現HSP90β結合在具感染力的EV71病毒顆粒上。藉由追蹤位在病毒顆粒上標有flag標定的HSP90β可以發現，病毒顆粒會攜帶這個HSP90β分子並一同感染至下一宿主中。當病毒顆粒上的HSP90β藉由HSP90功能抑制劑Geldanamycin抑制時，病毒的感染效率下降，但並不影響病毒進入宿主中的效率。而在EV71感染細胞內，HSP90能夠幫助病毒外殼蛋白(VP)的摺疊，當HSP90功能受抑制，未完整摺疊之病毒外套蛋白會經由蛋白脢體(proteasome)分解。藉由免疫共沉澱法與in vivo pull down可以發現HSP90β與病毒外殼蛋白具有交互作用。此外，HSP90α與HSP90β均與病毒3D、3CD蛋白具有交互作用。然而，當藉由siRNA抑制細胞中的HSP90β或大量表現HSP90β，感染細胞內之外殼蛋白並沒有因此明顯的下降或上升，可能是HSP90β與病毒3D病毒結合，調節病毒感染初期之複製。但觀察釋放出之具感染力的病毒顆粒，發現釋放之具感染病毒顆粒會因HSP90β表現量受抑制時而減少大約五倍，大量表現時而增加。藉由比較由受HSP90β siRNA抑制與scramble控制組所釋放之病毒顆粒密度，可以發現受HSP90β siRNA抑制之病毒顆粒密度較小。因此，HSP90β對於病毒顆粒的折疊、組裝與成熟扮演了很重要的色。
由以上結果得知，HSP90β在感染細胞內與HSP90α一同負向調節病毒複製;另一方面，HSP90β為幫助病毒外套蛋白正確摺疊之亞型，同時，HSP90β能幫助病毒組裝(assembly)，並成熟(maturation)為具感染力之病毒顆粒。在病毒成熟準備由宿主釋放時，HSP90β會被病毒顆粒攜帶，一同釋放至宿主外，感染至下一宿主中。

Enterovirus 71(EV71) is one of most important public health issue in Asia-pacific region including China, Taiwan, Singapore, and Malaysia. EV71 is major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. There is no vaccine or drug available and further research on virus-host interactions were needed. In the past, most of virus-host interaction research were focused on the interaction inside of cells, whereas little is concerns about the interaction outside of host cells. Here, we use the proteomic(LC/MS) approach to analysis the EV71 induced secretion proteins and identified one of these is HSP90β but not HSP90α . We further demonstrate the secretion HSP90β were associated with virus particle by sucrose density gradient, immunoprecipitation and observed by transmission electromicroscopy(TEM). By tracking the flag tag HSP90β on the virus particle, we found that the HSP90β was co-opt by virus particle and infected to next host. Geldanamucin(GA) , an HSP90 functional inhibitor, treatment on HSP90β associated virus particle reduced virus infectivity but without affecting EV71 entry.
Furthermore, in EV71 infected cell, hsp90 facilitate EV71 capsids protein folding and the unfolding capsid proteins were degraded by proteasome . The interaction between viral capsids protein and HSP90β were demonstrated by in vivo pull down and immunoprecipitation. The HSP90α and HSP90β also interacted with viral 3D、3CD protein. However, overexpression or silence of HSP90β didn’t show obvious increase or decrease in intracellular virus capsids protein level. These may cause by suppression of virus replication in the initial stage of infection by HSP90β. But the release virus titer were decrease about 5 fold when silence of HSP90βand increase when overexpression of HSP90β. These may indicated that the HSP90β is an important molecular for viral particle to proper folding and mature into infectious virus particle. Altogether, these result indicated that the HSP90β facilitating the proper folding of EV71 capsids, assembly and maturation of virus into infectious virus particle, and regulate virus replication with HSP90α by binding with viral 3D protein. After virus particle were assembly and release, the HSP90β were co-opt by virus particle and help the virus infected to next host.

指導教授推薦書 王永梁 博士
指導教授推薦書 Scott C. Schuyler 博士
口試委員會審定書
國家圖書館授權書
長庚大學圖書館授權書
誌謝………………………………………………………………………VI
英文摘要………………………………………………………………VIII
中文摘要………………………………………………………………IX
目錄………………………………………………………………………XI
圖目錄…………………………………………………………………XIV

第一章 簡介…………………………………………………………………………1
1. 腸病毒71型 (Enterovirus 71) 1
1-1. 腸病毒71行之特性與生活史 1
1-2. 腸病毒71行之臨床症狀與致病機制 3
2. 熱休克蛋白90 (Heat shock protein 90) 4
2-1. 熱休克蛋白90 4
2-2. 熱休克蛋白90之亞型 6
2-3. 熱休克蛋白90參與病毒感染 8
3. 研究動機 10
3-1. EV71感染細胞釋放蛋白可做為臨床應用與抗病毒標的並可能與病毒在宿主外的穩定性與感染力有關聯 10
3-2. 熱休克蛋白90α與β亞型(isoform)在受EV71感染之神經細胞中所扮演的角色 12
第二章 材料與方法…………………………………………………………………13
2-1. 細胞培養 (Cell culture) 13
2-2. 腸病毒71型感染與製備 (EV71 virus preparation) 14
2-3. 病毒效價測試 (Plaque assay) 14
2-4. 細胞存活率測試 (Cell viability) 15
2-5. SDS聚丙烯醯氨膠體分離法（sodium dodecyl sulfate polyacrylamide gel electrophoresis） 15
2-6. 西方墨點法(Western blotting) 16
2-7. 收集病毒感染細胞分泌培養液與分析(Virus infected cell secretion medium collection and analysis) 17
2-8. 銀染硝酸銀染色法 17
2-9. RNA萃取(RNA extraction) 18
2-10. RNA反轉錄(The reverse transcription of RNA) 19
2-11. 即時定量聚合脢反應(Real time Quantitative Polymerase Chain Reaction) ………………………………………………………………………………… 19
2-12. 轉染siRNA(Transfect siRNA) 20
2-13. 細胞表面病毒接受器中和試驗(Virus receptor neutralization assay) 21
2-14. 質體建構 (Construction) 21
2-15. 質體轉染 (Transfection of plasmid into Cell) 22
2-16. Geldanamycin與Lactacystin藥物處理(Geldanamycin and Lactacystin treatment) 23
2-17. 免疫共沉澱法(Co-Immunoprecipitation assay) 23
2-18. In vivo pull down 23
2-19. 穿透式電子顯微鏡(Transmission electron microscopic) 24
2-20. 蔗糖梯度離心(Sucrose density gradient) 24
2-21. 免疫螢光染色(Immunofluorescence) 25
第三章 實驗結果 27
3-1. 分析受腸病毒71型病毒感染神經膠質瘤細胞分泌蛋白 27
3-2. 受腸病毒71型、克沙奇病毒A16與Echo 病毒感染細胞分泌液中偵測到熱休克蛋白90β亞型 29
3-3. 釋放的熱休克蛋白90β與腸病毒71型病毒顆粒結合 32
3-4. 病毒顆粒上之HSP90β與病毒顆粒一同感染至下一宿主中 34
3-5. 釋放出的熱休克蛋白90β可能與病毒感染下一宿主有關 35
3-6. 熱休克蛋白90在感染細胞內參與在腸病毒71型結構性蛋白的摺疊 36
3-7. 熱休克蛋白90α與β亞型在細胞內的表現量 38
3-8. 熱休克蛋白90β與病毒蛋白3D、3CD與VP1有交互作用而HSP90α與病毒蛋白3D、3CD有交互作用 38
3-9. 熱休克蛋白90β亞型幫助病毒外套蛋白折疊、組裝、成熟為具感染力之病毒顆粒 ..39
3-10. 熱休克蛋白90α與90β亞型參予在病毒複製調解 41
第四章 討論 44
4-1. 在受EV71感染細胞分泌液中偵測到熱休克蛋白90β亞型 45
4-2. HSP90β與EV71病毒顆粒結合一同由宿主中釋出 48
4-3. HSP90β與病毒顆粒一同感染至下一宿主 50
4-4. 位於病毒顆粒上之HSP90β可能影響病毒感染率 50
4-5. 細胞內之HSP90能夠幫助病毒外套蛋白正確摺疊 51
4-6. 熱休克蛋白90α與β亞型在EV71感染細胞中可能扮演之角色 52
第五章 參考文獻 58
圖表 63
 
圖目錄
圖一、腸病毒71型在血清與無血清環境下之複製情形…..................63
圖二、細胞分泌培養液中之細胞骨架β-actin含量…………………64
圖三、受病毒感染後48小時細胞存活率…………………………….65
圖四、不同時間點之感染細胞分泌培養液中的總蛋白……………..66
圖五、比較受感染與未受感染細胞所分泌之蛋白差異……………..67
圖六、質譜分析結果…………………………………………………..68
圖七、利用西方墨點法分析受腸病毒71型病毒感染細胞HSP90α與HSP90β分泌量與細胞內之表現量……………………………………69
圖八、利用西方墨點發偵測RD細胞所分泌之HSP90β與分泌量..70
圖九、利用西方墨點法分析受Coxsackie virus A16與Echo virus病毒感染細胞HSP90α與HSP90β分泌量與細胞內之表現量……….71
圖十、受腸病毒71型感染細胞在不同時間點所分泌之HSP90β…72
圖十一、 受腸病毒71型感染細胞分泌液經20% sucrose cushion後無法偵測到albumin與peripherin…………………………………73
圖十二、利用蔗糖梯度密度離心分析腸病毒71型病毒顆粒與HSP90β是否位在同一個fraction中…………………………………………74
圖十三、利用免疫共沉澱法證明HSP90β與病毒顆粒間之結合…75
圖十四、以穿透式電子顯微鏡(TEM)觀察病毒顆粒與HSP90β位子.76
圖十五、當細胞表現腸病毒71型 replicon時HSP90β分泌量……77
圖十六、HSP90β與病毒顆粒一同釋放並感染至下一宿主………....78
圖十七、以GA抑制病毒顆粒上之HSP90β並以西方墨點法偵測病毒感染下一宿主之效率………………………………………………….79
圖十八、以GA抑制病毒顆粒上之HSP90β並以RT-qPCR偵測病毒進入細胞之效率……………………………………………………….80
圖十九、Geldanamycin對細胞存活率之影響……………………...81
圖二十、Geldanamycin對細胞內HSP90亞型表現之影響……….82
圖二十一、利用Geldanamycin處理腸病毒71型感染細胞對腸病毒71型病毒效價之影響………………………………………………....83
圖二十二、利用Geldanamycin處理腸病毒71型感染細胞對病毒蛋白表現量之影響……………………………………………………….84
圖二十三、當HSP90受到Geldanamycin抑制時，對於腸病毒71行進入到細胞中的影響………………………………………………….85
圖二十四、以Geldanamycin抑制感染細胞內HSP90之功能對於病毒複製之影響…………………………………………………………..86
圖二十五、檢測當HSP90功能受抑制時，未完整摺疊之病毒外套蛋白是否經由preotasome分解………………………………………….87
圖二十六、受腸病毒71型感染細胞中，HSP90α與HSP90β的表現量………………………………………………………………………..88
圖二十七、受腸病毒71型感染細胞中，HSP90α的RNA量……..89
圖二十八、以HSP90α與β抗體進行免疫共沉澱法，分析可能與HSP90α與β結合之病毒蛋白………………………………………..90
圖二十九、以3flag-HSP90α、3flag-HSP90β進行in vivo pull down assay偵測可能與HSP90α、HSP90β結合之宿主蛋白…………….91
圖三十、以免疫螢光染色確認HSP90β是否與病毒外套蛋白VP在感染細胞中位在同一位子上……………………………………………..92
圖三十一、以HSP90β siRNA抑制腸病毒71型感染細胞內之HSP90β表現量對於病毒外套蛋白摺疊之影響……………………………….93
圖三十二、以siRNA抑制感染細胞中的HSP90β表現對於對於腸病毒71型病毒顆粒釋放的影響…………………………………………94
圖三十三、在感染細胞內大量表現HSP90β對於腸病毒71型病毒顆粒釋放之影響…………………………………………………………..95
圖三十四、利用蔗糖密度梯度離心比較受 HSP90β siRNA抑制細胞內HSP90β表現之感染細胞與scramble RNA控制組感染細胞所分泌病毒顆粒之密度比較…………………………………………………..96
圖三十五、利用HSP90β抗體抑制細胞表面的HSP90β，以檢測HSP90β是否為腸病毒71型之受器(receptor)………………………97
圖三十六、抑制HSP90β對於腸病毒71型感染進入細胞之影響…98
圖三十七、以HSP90β siRNA抑制腸病毒71型感染細胞內之HSP90β表現量，對腸病毒71型病毒蛋白表現量之影響…………………….99
圖三十八、利用HSP90β siRNA抑制細胞內HSP90β表現量對於病毒複製效率之影響………………………………………………………100
圖三十九、大量表現帶有flag標定之HSP90β並偵測病毒蛋白表現情形………………………………………………………………………101
圖四十、大量表現帶有flag標定之HSP90α並偵測病毒蛋白表現情形………………………………………………………………………102
圖四十一、熱休克蛋白90在腸病毒71型生活史中所扮演之角色..103

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