臺灣博碩士論文加值系統

黃質病毒屬中的登革病毒（DEN）和日本腦炎病毒（JEV）在人類醫學上非常重要。Phosphatidylinositol 3-kinase (PI3K) 及它的下游Akt和調控許多不同的細胞功能有關（例如發育和細胞凋亡）。當日本腦炎病毒和登革病毒感染培養的細胞會在相當晚期的階段引發細胞凋亡，因此我們推論在黃質病毒感染時，PI3K/Akt的訊息傳遞可能扮演一定的角色。我們發現，在那些感染日本腦炎病毒和登革病毒的N18細胞中，磷酸化的Akt在感染早期有顯著的增加，而且此磷酸化形成和PI3K及lipid raft有關。透過加入PI3K的抑制劑LY294002或wortmannin抑制PI3K的活性，使得在感染的早期大幅提升病毒引起的細胞病變，但是對病毒顆粒的產生卻沒有影響。我們利用TUNEL以及caspase-3、poly(ADP-ribose) polymerase (PARP)的截切證實此嚴重細胞病變為細胞凋亡。利用pan-caspase、caspase-9及caspase-6的抑制劑而非使用caspase-8、caspase-3及caspase-7的抑制劑可以有效阻斷PARP的截切，因此推論在阻斷PI3K的訊息傳遞下，黃質病毒主要透過caspase-9和caspase-6引發細胞凋亡。此外，當PI3K活性被抑制時，大量表現Bcl-2於細胞中可以有效降低病毒引起的細胞凋亡，因此推測Bcl-2位於PI3K/Akt訊息傳遞的下游。總結我們的發現，我們的結果證明在日本腦炎病毒和登革病毒引發早期細胞自裁的路徑上，PI3K/Akt的訊息傳遞具有保護細胞免於早期死亡的功能。
日本腦炎病毒（JEV）和登革病毒二型（DEN-2）為一具有膜的病毒，它們藉由細胞膜上的接受器進行胞吞作用以及在低pH的情況下進行融合，然後使得病毒可以在細胞內膜進行複製。Lipid raft是由許多膽固醇所組成的膜結構，是一種具有許多細胞功能的平台。在這篇研究中，我們利用膽固醇拮抗劑（如methyl-- cyclodextrin和filipin III）來消耗膽固醇，結果發現膽固醇的消耗降低了日本腦炎病毒和登革病毒在細胞內的複製，並且也小幅影響病毒進入細胞的過程。透過膜漂浮分析，我們更進一步證明黃質病毒非結構性蛋白質和具有抵抗洗滌劑的膜架構同在一起，因此證明日本腦炎病毒和登革病毒的複製是位於lipid raft的膜結構上。有趣的是，我們也發現額外加入的膽固醇會抑制黃質病毒感染，與膽固醇提升Sindbis病毒的感染形成強大對比。膽固醇可能是藉由降低黃質病毒核醣核酸的脫離，而不是藉由干擾病毒細胞間的結合或進入來影響病毒感染細胞。膽固醇的抗病毒作用可能也發生在病毒進入細胞後的時期。我們的結果說明了在黃質病毒的生命週期中，對膜的組成，特別是膽固醇的數量，具有非常嚴格的要求。

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. As JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, as only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3 or -7 alleviated the levels of PARP cleavage following virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, as overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.
Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells via receptor-mediated endocytosis and low-pH-triggered membrane fusion, then replicate at intracellular membrane structures. Lipid rafts, the cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion of chelating with methyl--cyclodextrin or filipin III reduced JEV and DEN-2 infections mainly at the intracellular replication steps, and to a lesser extent on viral entry. By membrane flotation assay, we further demonstrated that several flaviviral nonstructural proteins were associated with detergent-resistant membrane structures, indicating the replication complex of JEV and DEN-2 are localized at the membranes possessing lipid raft property. Interestingly, we also found that extra amount of cholesterol readily blocked flaviviral infection, in great contrast to an alphavirus, sindbis virus whose infection was enhanced by cholesterol. Cholesterol might reduce the levels of viral RNA uncoating, but not at the initial infection steps of viral binding and entry. The anti-flaviviral effects of cholesterol might also take place intracellularly in the steps post viral adsorption. Our results thus suggest a stringent requirement of membrane components, especially the amounts of cholesterol for various steps of flavivirus life cycle.

CONTENTS
Pages
CHAPTER I: Flavivirus Activates Phosphatidylinositol 3-Kinase Signaling to Block Caspase-Dependent Apoptotic Cell Death at the Early Stage of Virus Infection 2
中文摘要 3
ABSTRACT 4
INTRODUCTION 5
MATERIALS AND METHODS 9
RESULTS 14
DISCUSSION 21
REFERENCES 27
FIGURES 34
CHAPTER II: The Roles of Cholesterol on Flavivirus Infection 43
中文摘要 44
ABSTRACT 45
INTRODUCTION 46
MATERIALS AND METHODS 50
RESULTS 55
DISCUSSION 62
REFERENCES 66
FIGURES 75

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