臺灣博碩士論文加值系統

干擾素（Interferon；簡稱 IFN）是一種非常有效的抗病毒細胞激素，在免疫系統中扮演著樞紐的角色。在本論文研究，我們發現當細胞感染同屬於黃質病毒屬的登革病毒與日本腦炎病毒時，細胞會因為病毒的複製以及新蛋白質的合成而產生乙型干擾素（IFN-beta）。我們探討其分子機轉發現：這兩種病毒所誘發乙型干擾素產生的過程中，都必須要活化NF-kappaB和干擾素調控因子（interferon regulatory factor；簡稱IRF），但是兩種病毒所活化的IRF種類不完全相同。當細胞中持續性表現的IRF-3被活化時，呈現在蛋白質電泳膠上的移動速率較慢，另外也會形成雙體複和物（dimer form），IRF-3在細胞中的分佈也會從細胞質轉進到細胞核。我們的實驗觀察到日本腦炎病毒造成上述之IRF-3的活化狀態比登革病毒明顯，但是登革病毒會誘發其他IRF，例如IRF-1、IRF-7的產生，日本腦炎卻不會。我們更進一步探討IRF-3和NF-kappaB的上游調控分子，證實retinoic acid inducible gene-I （RIG-I）和phosphatidylinosotol-3 kinase（PI3K）分別參與了黃質病毒所誘導的IRF-3和NF-kappaB之活化。因此，我們推論日本腦炎病毒與登革病毒藉由活化RIG-I/IRF-3和PI3K/NF-kappaB之訊息傳導機制來開啟宿主之先天性免疫反應。

In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and Dengue virus serotype 2 (DEN-2), leads to interferon-beta (IFN-beta) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-kappa B activation is essential for IFN-beta induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-kappa B activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-kappa B activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-kappa B signaling pathways.

中文摘要 4
ABSTRACT 5
KEYWORDS AND ABBREVIATIONS 6
CHAPTER ONE: INTRODUCTIOH 7
CHAPTER TWO: MATERIALS AND METHODS 14
1. Cells cultures and chemicals 15
2. Virus and viral infection 15
3. SDSPAGE and native PAGE immunoblot analysis 16
4. Plasmids 17
5. RT-PCR 20
6. Immunofluorescence assay 20
7. Luciferase reporter assay 21
CHAPTER THREE: RESULTS 22
1. JEV and DEN-2 infections induce IFN-beta expression 23
2. NF-kappa B activation is required for IFN-beta induction in virus-infected A549 cells 25
3. Different expression patterns of IRFs induced by JEV and DEN-2 infection 26
4. Regions of IRF-3 required for JEV activation 27
5. RIG-I is essential for flavivirus-induced IRF-3 activation 29
6. PI3K is involved in NF-kappaB activation and is required for IFN-beta induction by flavivirus infections 31
CHAPTER FOUR: DISCUSSION 33
CHAPTER FIVE: REFERENCES 41
APPENDIX: FIGURS 52
Fig. 1: JEV and DEN-2 infections induce IFN-beta gene expression 53
Fig. 2: Induction of IFN-beta by JEV and DEN-2 infections depends on viral replication and de novo protein synthesis 54
Fig. 3: NF-kB-dependent activation of IFN-b in JEV- and DEN-2-infected A549 cells 55
Fig. 4: Patterns of IRF expression by JEV and DEN-2 infections 57
Fig. 5: Regions and sequences required for IRF-3 activation by JEV infection 59
Fig. 6: JEV-induced IRF-3 and IFN- activation are independent of TBK-1 and IKK 60
Fig. 7: RIG-I is involved in JEV- and DEN-2-induced IRF-3 activation and IFN-beta promoter induction 63
Fig. 8: Role of PI3K signaling in the flavivirus-induced IFN-beta activation pathway 65
Fig. 9: Models of IFN induction and signaling pathway by JEV and DEN-2 infections 67

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