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研究生:原靜惠
研究生(外文):CHIN-HUEY GUAN
論文名稱:登革熱病毒非結構蛋白NS5核位訊息對病毒複製的影響及病毒非結構蛋白NS4A一適應性突變彌補NS5突變缺失之研究
論文名稱(外文):Effect of Dengue Virus NS5 Protein Nuclear Localization Sequence In Viral Replication And Ability of NS4A Adaptive Mutation to Compensate NS5 Mutations
指導教授:吳惠南
指導教授(外文):Huey-Nan Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:登革熱病毒
外文關鍵詞:Dengue Virus
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登革熱病毒屬於黃熱病毒科藉由病媒蚊子傳染給人類。病毒的基因體為約11 kb長之正股RNA,其可轉譯成一含約3000個胺基酸的多蛋白質,之後可被切割成三個結構蛋白質及七個非結構蛋白質。結構蛋白質負責包裹RNA以及組裝病毒顆粒子,而非結構蛋白則負責複製病毒基因體RNA。NS5是一個含900個胺基酸的多功能病毒非結構蛋白質,NS5蛋白質N端三分之一具有methyltransferase活性可參與病毒基因體RNA 5’ capping反應,而C端三分之二具有RNA-dependent RNA polymerase活性用於基因體RNA複製。NS5蛋白質第369至第389胺基酸區含有basic amino acid-rich clusters之核位訊息的保守型序列。在論文中我探討登革熱病毒NS5蛋白質核位訊息在NS5蛋白質進核、在活體外合成RNA的能力及在細胞內複製病毒基因體RNA所扮演的角色。為此,我分別將位在第371及372 (KK1)、第388及389 (KK2)或第371、372、388及389(KK12)的Lysine以Alanine取代。實驗得知野生型及分別帶有KK1、KK2或KK12的NS5重組蛋白質均具有類似之RNA-dependent RNA polymerase活性。登革熱病毒野生型複製子可在被轉染細胞中複製,當複製子之NS5蛋白質突變為KK1、KK2或KK12時會導致複製子喪失複製能力。在一罕見具有複製能力的NS5KK1複製子上發現一個NS4A蛋白質的突變(命名為NS4Am)。當我們把NS4Am引至NS5 KK1複製子後證實了NS4Am可顯著提升NS5KK1複製子的複製能力,然而NS4Am並無法彌補含有NS5KK2以及NS5KK12二突變複製子在複製能力上之缺失。由NS4Am可彌補NS5KK1突變之複製缺陷推測此二非結構蛋白質之間應具有交互作用。當NS5-綠螢光融合蛋白質單獨在細胞中表現時,絕大部分之融合蛋白質集中於細胞核,此進核能力會受到NS5之KK1與KK2突變而下降,更會因KK12突變而完全喪失。我發現當NS5蛋白質和其他病毒非結構蛋白質同時在細胞表現時,其進核效率比單獨表現的NS5蛋白質為低,NS4Am


突變蛋白質不改變野生型NS5蛋白質亦無法提升NS5 KK1突變蛋白質之進核的能力至野生型的程度。此結果顯示病毒基因體RNA之複製不全然受NS5蛋白質進核效率的調控。
Dengue virus (DENV) is an arthropod-borne human pathogen of the Flaviviridae family. DENV contains a ~11 kb positive-sense RNA genome encoding a polyprotein of ~3,000 amino acids. The viral polyprotein is processed into three structural proteins (C, prM and E) for viral RNA packaging and virion formation and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) for viral RNA replication. NS5 is a 900-amino acid protein with multiple functions: the N-terminal one third is a methyltransferase (MTase) participating in viral RNA 5’ capping, and the C-terminal two thirds is an RNA-dependent RNA polymerase (RdRp) responsible for viral RNA synthesis. The amino acids #369-389 of the NS5 RdRp domain contains basic amino acid rich clusters homologous to nuclear localization signal (NLS). In this report, I studied the role of the putative NLS of DENV NS5 in NS5 nuclear localization, NS5 RdRp activity in vitro, and viral replication in vivo. To these ends, I did lysine to alanine substitution at amino acid #371-372 (KK1), #388-389 (KK2), and both #371-372 and #388-389 (KK12) of NS5. Recombinant NS5 with KK1, KK2, or KK12 mutation was as active as their wild-type counterpart in catalyzing the RNA-dependent RNA synthesis in vitro. The wild-type DENV replicon but not the NS5 KK1, KK2, or KK12 mutant replicon replicated actively in cells transfected with replicon RNA. A second site NS4A mutation, name NS4Am, was identified in a replicating competent NS5 KK1 replicon. NS4Am partially compensated the replication defect of NS5 KK1 replicon when it was introduced into the NS5 mutant replicon, while it barely compensated the replication defect of NS5 KK2 or NS5 KK12 replicon. The finding of NS5 KK1 mutation being rescued by NS4Am suggests a genetic interaction between these two NS proteins. NS5-EGFP fusion protein located mainly at the nucleus. KK1 and KK2 mutations individually decreased the nuclear localization ability of the NS5-EGFP fusion protein, while the protein with KK12 mutation located exclusively in the cytoplasm. NS5 had lower nuclear localization efficiency when it was expressed with other NS proteins compared with the expression of NS5 alone. NS4Am did not alter the nuclear localization efficiency of wild-type NS5 neither did it elevate the nuclear localization efficiency of NS5 KK1 to the level of similar to that of the wild-type NS5. This result suggests that viral RNA replication is not simply regulated by the nuclear accumulation efficiency of NS5.
CHINESE ABSTRACT中文摘要 I
ENGLISH ABSTRACT IV

TABLE OF CONTENTS V
LIST OF TABLES VII
LIST OF FIGURES VIII
LIST OF APPENDIX IX
CHAPTER 1 - INTRODUCTION 1
CHAPTER 2 - MATERIALS AND METHODS 5
2.1 bacteria 5
2.2 Cell and cell culture 5
2.3 Plasmids 5
2.4 Site-directed mutagenesis to generate plasmid containing NS5 mutants 6
2.5 Plasmid construction 7
2.6 NS5 protein expression and purification 8
2.7 RdRp assay 9
2.8 RNA synthesis 9
2.9 RNA transfection and establish stable DENV replicon cell lines 9
2.10 DNA transfection and the determination of NS5 nuclear localization efficiency 10
2.11 Luciferase activity assay 11
2.12 Western blot analysis 12
2.13 Indirect immunofluorescence analysis (IFA) 12
2.14 RNA isolation and RT-PCR amplication 14
CHAPTER 3 - RESULTS 14
3.1 The effect of NS5 NLS mutation on the cellular distribution of NS5 14
3.2 In vitro RdRp assay of recombinant NS5 protein 15
3.3 Effect of NS5 NLS mutation on viral RNA replication 15
3.4 Identification and reconfirming of a NS4A mutation that rescued the replication defect of NS5KK1-NeoSGR 17
3.5 Confirmation of the genetic stability of DENV replicon in stable cell line 18
3.6 Effect of NS4Am on RNA replication 18
3.7 Effect of co-expression of NS4A on NS5 nuclear transportation 19
CHAPTER 4 - DISCUSSION 20
CHAPTER 5 - REFERENCES 23
CHAPTER 6 - TABLE 25
CHAPTER 7 - FIGURES 26
CHAPTER 8 - APPENDIX 48
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