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系統識別號 U0026-2407202001104600
論文名稱(中文) 含雙色胺酸功能區氧化還原酶在腸病毒A71型及單純皰疹病毒複製中的調控
論文名稱(英文) The control of enterovirus A71 and herpes simplex virus replication by WW domain-containing oxidoreductase
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 107
學期 2
出版年 108
研究生(中文) 陳亭羽
研究生(英文) Ting-Yu Chen
學號 T36064055
學位類別 碩士
語文別 英文
論文頁數 64頁
口試委員 指導教授-徐麗君
口試委員-詹明修
口試委員-王雅芳
中文關鍵字 腸病毒A71型  單純皰疹病毒  病毒複製  病毒生活史 
英文關鍵字 Enterovirus A71  Herpes simplex virus  virus replication  virus life cycle 
學科別分類
中文摘要 人類第16號染色體上容易脆裂的基因WWOX可以轉錄出一個命名為「含雙色胺酸功能區氧化還原酶」(WW domain-containing oxidoreductase,亦稱為WWOX)的腫瘤抑制蛋白質。WWOX除了有抑制腫瘤的功能之外,還被發現會調節胚胎發育、神經疾病、細胞凋亡以及病原體感染。腸病毒A71型是屬於小RNA病毒科中的腸病毒屬,我們實驗室先前的研究發現,WWOX蛋白質會抑制腸病毒A71型的感染。為了要更進一步了解WWOX抑制病毒感染的機制,我們選擇了第一型人類皰疹病毒此DNA病毒作為另一個研究模式,試圖比較DNA病毒與腸病毒A71型RNA病毒的感染有何異同。我們的實驗發現,缺乏Wwox基因的小鼠在利用腹腔注射方式感染人類皰疹病毒後,小鼠的生存率與體重增加幅度都明顯比對照組低。綜合以上結果發現,WWOX不只會抑制腸病毒A71型,也會抑制人類皰疹病毒的感染。病毒在感染宿主細胞後,會劫持宿主細胞的資源幫助病毒複製。為了探究WWOX如何抑制病毒感染,我們進一步偵測病毒蛋白質表現量,發現在細胞感染腸病毒A71型後,WWOX會抑制病毒蛋白質在宿主細胞的表現,進一步抑制病毒複製。未來,我們將針對WWOX如何透過調節宿主細胞內的作用,來抑制腸病毒A71型感染的機制進行更深入的探討。
英文摘要 Fragile WWOX gene encodes a tumor suppressor WW domain-containing oxidoreductase. In addition to its suppression of cancer progression, WWOX protein has been reported to regulate embryonic development, neuronal diseases, apoptosis and pathogenic infections. Enterovirus A71 (EV-A71) is an RNA virus that belongs to Enterovirus genus in Picornavirus family. Previous studies in our laboratory showed that WWOX suppressed EV-A71 infection. To study whether WWOX also suppresses DNA virus infection, herpes simplex virus (HSV) was tested in this study. Our results showed that Wwox-/- mice had a higher mortality rate and body weight loss as compared with Wwox+/+ and Wwox+/- littermates after intraperitoneal injection of HSV-1. Together, our results suggest that WWOX suppresses both EV-A71 and HSV-1 replication in host cells. Viruses usually hijack host cell machinery to ensure viral genome replication and protein synthesis. To investigate the molecular mechanism by which WWOX downregulates virus replication, we first examined the expression of viral proteins after EV-A71 infection in WWOX-overexpression or -knockdown cell lines. As compared with the control cells, the expression levels of EV-A71 proteins were decreased in the RD cells overexpressing WWOX. In the future, the molecular mechanism by which WWOX suppresses viral replication via regulating signaling pathways in host cells will be further investigated.
論文目次 摘要 I
Abstract III
Acknowledgements V
Contents VII
Figure Index XI
Introduction 1
Enterovirus A71 (EV-A71) infection 1
EV-A71 life cycle 1
The viral protein functions during EV-A71 replication 2
Herpes simplex virus 1 (HSV-1) infection 2
HSV-1 life cycle 3
Host factors involved in virus infection 3
ER stress and unfolded protein response (UPR) 4
EV-A71 replication and UPR induction 5
The regulation of EV-A71 replication by host factors 5
WW domain-containing oxidoreductase (WWOX) 6
The role of WWOX in EV-A71 infection 6
Materials and Methods 8
A. Materials 8
A-1 Cell lines 8
A-2 Virus 8
A-3 Plasmid construction 8
A-4 Reagents and chemicals 8
A-5 Kits 10
A-6 Antibodies 10
A-7 shRNA clones 12
A-8 PCR primers 12
A-9 Consumables 12
A-10 Instruments 13
B. Methods 15
B-1 Cell culture 15
B-2 Generation of EV-A71 and HSV-1 15
B-3 Virus infection of mice 16
B-4 Plaque assay 16
B-5 Transfection of WWOX shRNA 17
B-6 Transfection of viral protein by electroporation 18
B-7 RNA extraction 18
B-8 Reverse transcription (RT) 20
B-9 Polymerase chain reaction (PCR) 21
B-10 Protein extraction 23
B-11 Membrane protein extraction 25
B-12 Protein quantification 26
B-13 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 26
B-14 Plasmid DNA purification 29
B-15 Immunofluorescence (IF) 31
Results 33
WWOX suppresses EV-A71 and HSV-1 infections 33
WWOX decreases viral protein expression and viral production, but not HSV-1 RNA levels, in HSV-1-infected RD cells 33
WWOX suppresses the expression of EV-A71 replication-related proteins 2B, 2C, 3AB and 3CD at ER membrane 34
WWOX suppresses the targeting of EV-A71 2B to the membranous structure in host cells 35
WWOX suppresses the expression of EV-A71 replication-related protein 2B at ER during EV-A71 initial translation and replication complex formation 35
WWOX suppresses ER remodeling after EV-A71 infection 36
WWOX increases UPR pathways after EV-A71 infection 37
WWOX downregulates UPR pathways during EV-A71 initial translation 37
Discussion 39
The roles of WWOX in EV-A71 and HSV-1 infections and neurological pathogenesis 39
Viruses may differentially regulate three UPR pathways 40
Viruses can still trigger IRES-mediated viral protein translation upon PERK-eIF2α pathway activation 40
Conclusion 42
References 43
Figures 47
Figure 1 47
Figure 2 48
Figure 3 50
Figure 4 53
Figure 5 54
Figure 6 55
Figure 7 56
Figure 8 57
Figure 9 58
Figure 10 59
Figure 11 60
Appendix 61
Appendix I 61
Appendix II 62
Appendix III 63
Appendix IV 64
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