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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉秀慧 | zh_TW |
dc.contributor.advisor | Shiou-Hwei Yeh | en |
dc.contributor.author | 謝季伶 | zh_TW |
dc.contributor.author | Chi-Ling Hsieh | en |
dc.date.accessioned | 2021-07-11T15:43:23Z | - |
dc.date.available | 2024-02-28 | - |
dc.date.copyright | 2018-10-09 | - |
dc.date.issued | 2018 | - |
dc.date.submitted | 2002-01-01 | - |
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Loeb, The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication. J Virol, 2011. 85(3): p. 1298-309. 44. Jung, J., et al., 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): p. 8754-67. 45. Albin, C. and W.S. Robinson, Protein kinase activity in hepatitis B virus. J Virol, 1980. 34(1): p. 297-302. 46. Gerlich, W.H., et al., Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol, 1982. 42(3): p. 761-6. 47. Perlman, D.H., et al., Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids. Proc Natl Acad Sci U S A, 2005. 102(25): p. 9020-5. 48. Pugh, J., A. Zweidler, and J. Summers, Characterization of the major duck hepatitis B virus core particle protein. 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Ludgate, L., et al., Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids. J Virol, 2012. 86(22): p. 12237-50. 78. Ludgate, L., et al., Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation. J Virol, 2016. 90(12): p. 5830-5844. | - |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79093 | - |
dc.description.abstract | B型肝炎病毒(Hepatitis B virus;HBV) 所產生的核殼蛋白(HBV core protein;HBc)是由183個胺基酸組成,其中1-149個胺基酸(N-terminal domain;NTD) 為assemble domain,負責病毒核殼(nucleocapsid)的組裝;而150-183個胺基酸(C-terminal domain;CTD) 為nucleic acid-binding domain,負責病毒pgRNA之包裹(encapsidation)。HBc為一磷酸化蛋白,磷酸化主要發生在CTD中的絲氨酸(Serine)位點。先前研究通過絲氨酸突變為丙氨酸(Alanine),模擬去磷酸化形式HBc,發現Ser155,Ser162,Ser170的磷酸化修飾會參與HBV RNA encapsidation以及DNA synthesis等步驟之調控。然而CTD的磷酸化在wild type HBV複製子中的作用以及潛在的機制仍有待釐清。
在本研究中,我們使用C-S170抗體以檢查不同複製時期的HBc-S170磷酸化,C-S170抗體可專一性識別含有去磷酸化Ser170的HBc。通過引入特定的基因突變在不同階段停止病毒複製,且不影響HBc蛋白密碼子。結果顯示HBc-S170在複製週期中磷酸化呈動態改變,其在pgRNA encapsidation之前為高度磷酸化;而大多數含有核酸的核殼(genome-containing nucleocapsids)為去磷酸化。此外根據在HBc-S170位點不能去磷酸化的HBV突變體中,我們發現pgRNA encapsidation顯著降低,這顯示HBc-S170的去磷酸化對於encapsidation可能是重要的。而通過磷酸酶抑制劑處理下,nucleocapsid所包裹的pgRNA明顯減少,進一步證實了這種可能性。由於在S170A突變體複製子中鑑定出其會降低pgRNA encapsidation,由此可知S170A的磷酸化對於RNA衣殼化之前的某些特定事件也是重要的,目前正在研究中。 | zh_TW |
dc.description.abstract | Hepatitis B virus (HBV) core protein consists of 183 amino acids, in which 1-149 amino acids (N-terminal domain; NTD) is responsible for the assembly of nucleocapsids and 150-183 amino acids (C-terminal domain; CTD) is responsible for the encapsidation of the viral pregenomic RNA (pgRNA). HBc is a phosphorylated protein, with the phosphorylation sites at Serine residues in CTD. Approached by mutation of Serine to Alanine, mimicking the dephosphorylated form, phosphorylation of Ser155, Ser162, Ser170 has been suggested to participate in the regulation of viral RNA encapsidation and DNA synthesis process. However, the effects in wild-type replicon and also the underlying mechanism are still remained to be addressed.
In this study, we used the C-S170 antibody, which specifically recognizes the HBc epitope containing dephosphorylated Ser170, to examine the HBc-S170 phosphorylation pattern of different replication intermediates. The viral replication is stopped at different stages by introducing specific genetic mutations, which however did not affect the HBc protein codon. The results showed a dynamic phosphorylation of HBc-S170 through the replication cycle, which was highly phosphorylated before pgRNA encapsidation and most of the genome-containing nucleocapsids were dephosphorylated. With the evidence from an HBV mutant which could not be dephosphorylated at HBc-S170, we found that pgRNA encapsidation is significantly decreased. It suggested that dephosphorylating of HBc-S170 could be important for encapsidation. This possibility has been further validated by a decrease of pgRNA encapsidated under the treatment of phosphatase inhibitor. However in the previous study, a decreased RNA encapsidation was identified in the S170A mutant replicon, phosphorylation of S170A could be also important for the certain specific event before RNA encapsidation, which is currently under investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:43:23Z (GMT). No. of bitstreams: 1 ntu-107-R05445130-1.pdf: 2041656 bytes, checksum: 41be8fd71117d2e5630d67fd20c085ab (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝 I
摘要 II Abstract III 目錄 IV 圖表目錄 VI 第一章 序論 1 1.1 HBV簡介 1 1.2 HBV生活史 2 1.3 HBV核殼蛋白之結構與功能 3 1.4 HBV核殼蛋白磷酸化修飾對病毒生活史之影響 4 第二章 研究假說與策略 6 第三章 實驗材料與方法 7 3.1 質體 7 3.2 細胞培養 (Cell culture) 9 3.3 細胞轉染(Transfection) 10 3.4 蛋白質抽取 10 3.5 純化 HBV nucleocapsid 內之核酸 10 3.6 去磷酸化反應 (Lambda Protein Phosphatase reaction) 11 3.7 西方墨點法(Western blot) 11 3.8 HBV capsid之偵測 11 3.9 北方墨點法 (Northern blot) 12 3.10 南方墨點法 (Southern blot) 12 第四章 結果 14 4.1 專一性辨認B型肝炎病毒core protein的Ser170位點去磷酸化修飾之抗體 14 4.2 B型肝炎病毒在不同的複製階段中core protein的Ser170位點磷酸化程度 15 4-3 Ser170位點去磷酸化之HBc protein影響HBV RNA encapsidation 17 4.4 Ser170位點磷酸化之core protein影響capsid的組成 19 4.5 core protein磷酸化與去磷酸化對B型肝炎病毒的影響 20 第五章 討論 21 參考文獻 24 圖表 32 | - |
dc.language.iso | zh_TW | - |
dc.title | 探討B型肝炎病毒核殼蛋白C端之Serine 170位點磷酸化修飾在HBV生活史之影響 | zh_TW |
dc.title | Phosphorylation of C-terminal Domain Serine-170 of Hepatitis B Virus Core Protein in Regulating the Viral Life Cycle | en |
dc.type | Thesis | - |
dc.date.schoolyear | 106-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 陳培哲;鄧述諄;王聖涵 | zh_TW |
dc.contributor.oralexamcommittee | Pei-Jer Chen;Shu-Chun Teng;Sheng-Han Wang | en |
dc.subject.keyword | B型肝炎病毒,核殼蛋白,磷酸化,核酸包裹, | zh_TW |
dc.subject.keyword | hepatitis B virus,nucleocapsid protein,phosphorylation,encapsidation, | en |
dc.relation.page | 46 | - |
dc.identifier.doi | 10.6342/NTU201802669 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2018-08-10 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 微生物學研究所 | - |
dc.date.embargo-lift | 2023-10-09 | - |
顯示於系所單位: | 微生物學科所 |
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