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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉秀慧 | |
| dc.contributor.author | YU-JIE HE | en |
| dc.contributor.author | 何宇傑 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:10:00Z | - |
| dc.date.available | 2024-08-28 | |
| dc.date.copyright | 2019-08-28 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-09 | |
| dc.identifier.citation | 1. Liang TJ. Hepatitis B: The Virus and Disease. Hepatology. 2009;49(5):S13-S21.
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Phosphorylation and Nuclear-Localization of the Hepatitis-B Virus Core Protein - Significance of Serine in the 3 Repeated Sprrr Motifs. J Virol. 1995;69(2):1025-9. 38. Jung J, Hwang SG, Chwae YJ, Park S, Shin HJ, Kim K. Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication. J Virol. 2014;88(16):8754-67. 39. Zhao Q, Hu ZY, Cheng JJ, Wu S, Luo Y, Chang JH, et al. Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation. J Virol. 2018;92(13). 40. Gazina EV, Fielding JE, Lin B, Anderson DA. Core protein phosphorylation modulates pregenomic RNA encapsidation to different extents in human and duck hepatitis B viruses. J Virol. 2000;74(10):4721-8. 41. Lan YT, Li J, Liao WY, Ou JH. Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication. Virology. 1999;259(2):342-8. 42. 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Hepatitis B virus DNA splicing in Lebanese blood donors and genotype A to E strains: implications for hepatitis B virus DNA quantification and infectivity. J Clin Microbiol. 2012;50(10):3159-67. 53. Rosmorduc O, Petit MA, Pol S, Capel F, Bortolotti F, Berthelot P, et al. In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA. Hepatology. 1995;22(1):10-9. 54. Terre S, Petit MA, Brechot C. Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo. J Virol. 1991;65(10):5539-43. 55. Gunther S, Sommer G, Iwanska A, Will H. Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA. Virology. 1997;238(2):363-71. 56. Redelsperger F, Lekbaby B, Mandouri Y, Giang E, Duriez M, Desire N, et al. Production of hepatitis B defective particles is dependent on liver status. Virology. 2012;431(1-2):21-8. 57. Bayliss J, Lim L, Thompson AJ, Desmond P, Angus P, Locarnini S, et al. Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma. J Hepatol. 2013;59(5):1022-8. 58. Chen J, Wu M, Wang F, Zhang W, Wang W, Zhang X, et al. Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy. Sci Rep. 2015;5:16459. 59. Soussan P, Garreau F, Zylberberg H, Ferray C, Brechot C, Kremsdorf D. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA. J Clin Invest. 2000;105(1):55-60. 60. Soussan P, Tuveri R, Nalpas B, Garreau F, Zavala F, Masson A, et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J Hepatol. 2003;38(3):343-8. 61. Chen WN, Chen JY, Jiao BY, Lin WS, Wu YL, Liu LL, et al. Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78653 | - |
| dc.description.abstract | 乙型肝炎病毒 (Hepatitis B virus，HBV) 的pregenomic RNA (pgRNA) 除了會轉譯出聚合酶和核殼蛋白 (HBV core protein; HBc) 之外，也會經由剪接(splicing) 所產生的spliced RNAs (spRNAs) 製造出數種病毒蛋白質。在眾多spRNAs中，SP1 spRNA為表現量最多之一員，會轉譯出相較HBc183缺少最後一個胺基酸Cys183之HBc-Cys蛋白質。HBc結構主要可分為兩個domain: 第1-149胺基酸 (N-terminal domain; NTD)負責nucleocapsid的組裝，第150-183胺基酸 (C-terminal domain; CTD)則負責病毒核酸的包裹。由於HBc183與HBc-Cys除了CTD尾端之Cys外，NTD和CTD胺基酸序列完全相同，因此我們提出一個假說是HBc183與HBc-Cys可以一起組裝形成capsid，但在HBV的生活史中各司其職具備不同的功能。
藉由來自濱松大學 (Hamamatsu University) Suzuki教授所提供的HBc-Cys 專一性抗體，我們發現HBc-Cys存在於sucrose gradient 之capsid fraction中，顯示了HBc-Cys會與HBc183一同組裝形成病毒capsid。此外，我們也利用只表現HBc183或HBc-Cys的replicon來研究這兩個蛋白質在capsid組裝的過程中是否有所差異，結果發現HBc183之Cys183參與形成由雙硫鍵鍵結的核殼蛋白dimer結構。然而在CMV-WT和缺少SP1的CMV-A487C replicon所轉染的細胞中，我們觀察到兩者之間無論是在capsid組裝過程、DNA複製和病毒顆粒的釋放皆沒有顯著差異。而同樣的實驗也在貼近正常HBV病毒複製過程的AAV-HBV replicon系統中進行，以進一步探究HBc183或HBc-Cys在病毒複製過程，尤其是capsid的包膜 (envelopment) 和病毒顆粒釋放是否存在差異。本論文研究結果將有機會解開HBc183與HBc-Cys各自在HBV生活史中所扮演的功能性角色。 | zh_TW |
| dc.description.abstract | In addition to encode the polymerase and core (HBc) protein, the pregenomic RNA (pgRNA) of hepatitis B virus (HBV) also produces several viral proteins encoded by the pgRNA derived spliced RNAs (spRNAs). Among them, HBc-Cys encoded by the most abundant SP1 spRNA consists of same protein sequence with HBc but lacks only one cysteine at the very C terminal. As documented, HBc protein contains two functional domains: the N terminal domain (NTD, 1-149 a.a., responsible for nucleocapsid assembly) and the C-termianl domain (CTD, 150-183 a.a., responsible for packaging viral RNA). As HBc and HBc-Cys share the same NTD and CTD, we proposed a hypothesis that both HBc and HBc-Cys can be packaged into the viral capsids, but with different functions in viral replication cycle.
Aided by the HBc-Cys-specific antibody kindly provided by Prof. Suzuki (Hamamatsu University), we first found the presence of HBc-Cys protein in the sucrose gradient fractions enriched of viral capsids. It thus suggested the assembly of HBc-Cys together with HBc into the capsids. HBV replicon constructs, expressing either HBc183 or HBc-Cys alone, were then used to examine any difference between HBc and HBc-Cys in capsid assembly process. We found the Cys183 in HBc is involed in the formation of disulfide-bond mediated dimers, the basic subunit of viral capsids. The putative function of HBc183 or HBc-Cys in viral replication cycle has been examined in CMV-WT and SP1 deficient HBV replicons, which however did not show any significant difference for capsid assembly, DNA synthesis and virion release. The same experiment has been applied to the AAV-HBV replicons, which can help examine the envelopment and virion secretion process. We expect the results can help unravel the distinct functions of HBc183 and HBc-Cys in viral replication cycle. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:10:00Z (GMT). No. of bitstreams: 1 ntu-108-R06445104-1.pdf: 2404058 bytes, checksum: 5e4da08231423e0e21fa4109f6b47705 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 誌謝 1
摘要 2 Abstract 3 圖表目錄 6 第一章 序論 7 1.1 Hepatitis B virus (HBV)簡介 7 1.2 HBV 生活史 8 1.3 HBV核殼蛋白結構與功能 9 1.4 HBV RNA剪接(Splicing) 10 1.5 SP1 spRNA轉譯的HBc-Cys蛋白質介紹 11 第二章 研究假說與策略 13 第三章 實驗材料與方法 14 1. 質體建構 14 2. Site-direct mutagenesis 16 3. 細胞培養 17 4. 細胞轉染 17 5. 蛋白質抽取 18 6. 細胞培養液之Cushion實驗 18 7. Sucrose gradient分析 18 8. 西方墨點法分析 19 9. HBV capsid之偵測 19 10. HBV Particle gel 分析 19 11. 純化HBV nucleocapsid內之RNA 20 12. 北方墨點法分析 21 第四章、實驗結果 22 4.1 建構只表現HBc及HBc-Csy的HBV replicon之相關質體 22 4.2 探討HBV Capsid中是否含有HBc-Cys蛋白 23 4.3 HBc183而非HBc-Cys會形成由雙硫鍵鍵結的Dimer結構 24 4.4 HBc利用Cys183與Cys183形成雙硫鍵鍵結的Dimer結構 25 4.5 HBc183會與HBc-Cys 交互作用組裝形成capsid結構 26 4.6 缺少HBc-Cys對 CMV-HBV replicon於細胞內組裝複製及病毒顆粒釋放無顯著影響 26 4.7 缺少HBc-Cys對 AAV-HBV replicon於細胞內組裝複製無顯著影響 27 4.8 AAV-WT及AAV-A487C replicon釋放至細胞外病毒顆粒之比較 28 第五章 討論 29 參考文獻 32 圖表 41 | |
| dc.language.iso | zh-TW | |
| dc.subject | RNA剪接 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | 組裝 | zh_TW |
| dc.subject | dimerization | zh_TW |
| dc.subject | 核殼蛋白 | zh_TW |
| dc.subject | assembly | en |
| dc.subject | hepatitis B virus | en |
| dc.subject | dimerization | en |
| dc.subject | nucleocapsid | en |
| dc.subject | RNA splicing | en |
| dc.title | 探討B型肝炎病毒核殼相關蛋白質對病毒複製過程之影響 | zh_TW |
| dc.title | Hepatitis B Virus Capsid Proteins Involved in Viral Replication Cycle | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳培哲,鄧述諄 | |
| dc.subject.keyword | B型肝炎病毒,組裝,dimerization,核殼蛋白,RNA剪接, | zh_TW |
| dc.subject.keyword | hepatitis B virus,assembly,dimerization,nucleocapsid,RNA splicing, | en |
| dc.relation.page | 54 | |
| dc.identifier.doi | 10.6342/NTU201902965 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-08-28 | - |
| 顯示於系所單位： | 微生物學科所 | |
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