日本腦炎病毒(Japanese encephalitis Virus，簡稱JEV)屬於黃質屬的病毒，是引起人類腦炎的主要因子。病毒需利用許多宿主蛋白質以完成其生活史，本實驗室之前研究顯示，利用蛋白質體表現分析時發現在日本腦炎病毒感染的宿主細胞中，熱休克蛋白質60 (Heat shock protein 60，簡稱HSP60)表現量會下降，其在JEV生活史中扮演何種功能仍屬未知。本論文進一步利用西方轉印法證明日本腦炎病毒感染確實會抑制HSP60表現。此外，過量表現HSP60會幫助病毒NS5蛋白質的轉譯，而利用基因靜默法抑制HSP60表現時則會抑制病毒NS5蛋白質的表現。分析病毒的力價，發現不論增加或降低NS5蛋白質的表現對於病毒力價皆未有顯著影響。細胞存活率實驗分析結果發現，抑制HSP60表現的細胞在病毒感染後之存活率顯著下降，證明HSP60對細胞存活有其必要性。利用免疫螢光染色法證明JEV感染會引致細胞質內部分之HSP60進入細胞核。同時HSP60和病毒蛋白質NS5及prM/E有共定位的現象。利用3D活細胞顯微鏡觀察病毒感染活細胞的胞器形態，發現在抑制HSP60表現的細胞，於病毒感染後會出現較短小的粒線體，並減少病毒感染引起合胞體的數量。總括而言，本論文結果說明宿主蛋白質HSP60在日本腦炎病毒生活史中扮演重要的功能。

Japanese Encephalitis Virus (JEV) belongs to Family of flaviviridae and is a causative agent for human encephalitis. Completion of viral life cycle relies on host factors. Previous studies in our laboratory have shown the reduction of host heat shock protein 60 (HSP60) in JEV-infected cells using proteomics methods. However, function of HSP60 involved in JEV life cycle remains unclear. In this study, the reduction of HSP60 was further verified using western blot analysis. Overexpression of HSP60 in JEV-infected cells led to an increase of viral non-structural protein 5 (NS5, encoding RNA-dependent RNA polymerase). Accordingly, knocking down of HSP60 impaired viral NS5 translation. Elevation or reduction of NS5 proteins has no significant effect on virus titers. Knocking down of HSP60 in JEV-infected cells significantly reduced survival cells indicating that HSP60 is important for cell viability. Immunofluorescence assay showed that infection of JEV promotes HSP60 nuclear localization. Furthermore, HSP60 colocalized with viral NS5, and structural proteins (prM/E) in JEV-infected cells. Subcellular morphology change including shorter dynamics mitochondria and less JEV-induced syncytia were observed in the HSP60-knockdown cells under Nanolive’s 3D live cell microscope. Taken together, these results provide new insights of HSP60 into virus-host interaction in JEV life cycle.

Chapter 1. INTRODUCTION 1
1. Japanese encephalitis 1
2. Japanese encephalitis virus genome 1
3. Flavivirus life cycle 2
4. Identification of heat shock protein 60 4
5. Aim of this study 6
Chapter 2. MATERIAL AND METHODS 7
1. Cells lines and virus strain 7
2. Preparation of virus stocks 7
3. Transfection 8
4. Western blotting 8
5. RNA interference 10
6. Plaque assay 10
7. Immunofluorescence assay 11
8. Cell morphology assay 12
9. Cell viability assay 12
Chapter 3. RESULTS 15
1. JEV infection caused downregulation of HSP60 proteins 15
2. Overexpression of recombinant HSP60 facilitates NS5 translation 15
3. Knocking down HSP60 expression impairs NS5 translation 17
4. HSP60 plays a role in cell survival upon JEV infection
18
5. HSP60 redistributed into nucleus and colocalized with viral proteins upon JEV infection 19
6. Downregulation of HSP60 enhances JEV induced mitochondrial damage 19
Chapter 4 DISCUSSION
1. HSPs play crucial roles for viral life cycle 21
2. 2. JEV infection or knocking down of HSP60 induces cell death 22
3. JEV infection causes redistribution of HSP60 in the infected cells 23
4. JEV infection downregulates HSP60 and induces mitochondrial damage 24
Chapter 5. REFERENCES 27
Tables 30
Figures 31

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