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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 施嘉和(Chiaho Shih) | |
| dc.contributor.author | Chih-Hsu Chang | en |
| dc.contributor.author | 張志旭 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:46:08Z | - |
| dc.date.available | 2022-02-15 | |
| dc.date.available | 2022-11-24T03:46:08Z | - |
| dc.date.copyright | 2022-02-15 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-01-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81371 | - |
| dc.description.abstract | 在目前B型肝炎病毒的研究，其病毒顆粒的細胞外分泌機制依然需要進一步地探討。而在我們的研究中探討B型肝炎病毒的反轉錄酶突變株，其突變點為反轉錄酶胺基酸序列中YMDD模體（motif）置換為YMHA，而我們發現到此病毒突變株YMHA在培養中無法分泌病毒顆粒至培養液中，其原因可能為病毒聚合酶基因與封套膜基因的開放閱讀框架重疊，而導致其突變亦同時發生於封套膜蛋白的小環狀結構（small loop）上，而致使無法分泌病毒顆粒。但在過去的研究中指出封套膜蛋白的小環狀結構僅會影響D型肝炎病毒的病毒顆粒分泌，而非B型肝炎病毒的病毒顆粒分泌。因此，在此研究中我們想透過點突變技術（site-directed mutagenesis）以及瓊脂糖凝膠電泳（native agarose gel electrophoresis）來重新探討小環狀結構與B型肝炎病毒的病毒顆粒分泌之間的關係。我們發現到小環狀結構上胺基酸位點196或是位點198將其置換成脯胺酸（proline）後，會同時無法分泌病毒顆粒以及無基因組的病毒顆粒（genome-free virion），而22 nm亞病毒顆粒(subviral particles)的分泌則不受到影響。令人意外地，當胺基酸位點196置換成白胺酸（leucine）後，則會提升病毒顆粒的分泌。然而，在重要兩個胺基酸位點196以及198中間的位點197置換成脯胺酸後，則不影響病毒顆粒的分泌。透過表現不同的野生株封套膜蛋白的互補方法來分析小環狀結構突變株M198P，我們發現單獨表現野生株小型封套膜蛋白即可以大幅度地回復病毒顆粒的分泌。最後，我們了解到小型封套膜蛋白上小環狀結構突變對於病毒顆粒的分泌具有位點特異性。因此，綜觀以上有趣的現象，小環狀結構對於病毒顆粒型態發生及分泌，值得我們進一步地探討其所扮演微妙的角色。 | zh_TW |
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| dc.description.tableofcontents | Thesis Approval Form ii 誌謝 iii 中文摘要 iv Abstract v Table of Contents vii Chapter 1 - Introduction 1 1.1 Overview of Hepatitis B virus 2 1.2 Genome and structure of HBV 2 1.3 HBV life cycle 5 1.4 Morphogenesis and secretion of viral particles 6 1.5 HBV/Hepatitis D virus (HDV) infection 8 1.6 Cytosolic loops of envelope protein for HDV and HBV 9 1.7 Aim of study 10 Chapter 2 - Materials and methods 12 2.1 Cell line and transfection 13 2.2 Plasmids 13 2.3 Native agarose gel electrophoresis for western and southern blot analysis 14 2.4 Immunofluorescence analysis (IFA) 15 2.5 Immunoprecipitation and western blot assay 15 2.6 Protein topology prediction 16 2.7 Quantification of HBV virion DNA 16 2.8 Northern blot analysis 17 Chapter 3 - Results 18 3.1 No virion secretion of polymerase mutant YMHA 19 3.2 Polymerase mutant YMHA contains concurrent envelope mutations 19 3.3 Cytoplasmic distribution of HBsAg 20 3.4 Core-envelope interactions 21 3.5 A nuanced position effect in the small loop on genome-containing virion secretion 21 3.6 Drug-resistant polymerase mutant YIDD contains envelope mutations W196L/S 23 3.7 The small loop from the small S envelope is important for virion secretion 23 Chapter 4 – Discussion and conclusion 25 4.1 A nuanced role of the small loop in virion morphogenesis and secretion 26 4.2 Core-envelope interaction in virion secretion 27 4.3 L and S envelope proteins in virion assembly and secretion 28 4.4 Drug resistant HBV polymerase mutants in virion secretion 29 4.5 ESCRT Machinery in HBV virion secretion 30 4.6 The host factors involved in small loop of small envelope proteins 31 4.7 Conclusion 31 Chapter 5 – References 32 Chapter 6 – Figures 48 Fig. 1 A The predicted topology of the folded HBV envelope protein on the ER membrane contains a cytosolic large loop CYL-I and a small loop CYL-II 49 Fig.2 A motif YMDD in the wild type reverse transcriptase (RT) domain has been engineered into a YMHA motif 50 Fig. 3 HBV virion secretion is blocked by mutations within a cytosolic small loop of the envelope protein (HBsAg) 51 Fig. 4 Intracellular distribution of HBs protein in the cytoplasm by IFA 52 Fig. 5 Similar core-envelope interactions were detected between wild type and small loop mutants in HuH7 cells by immunoprecipitation assay and Western blot analysis 53 Fig. 6 Similar core-envelope interactions were detected between wild type and small loop mutants in HepG2-NTCP cells by immunoprecipitation assay and Western blot analysis 54 Fig. 7 Secretion of genome-containing virions is also affected by proline substitution in the small loop at amino acid 196–198 in a position-dependent manner 55 Fig. 8 Virion-associated HBV DNA genome of WT and small loop mutants in the supernatant of HuH-7 cells was analyzed by qPCR 57 Fig. 9 Two lamivudine-resistant polymerase mutants are normal in virion secretion 58 Fig. 10 Virion-associated HBV DNA from WT and Mutants were analyzed by qPCR as described in Fig. 8 59 Fig. 11 Schematic representation of envelope protein 60 Fig. 12 Validation of envelope protein expression vector 61 Fig. 13 Only the small envelope protein can rescue the virion secretion defect of the proline substitution mutant M198P 62 Fig. 14 Virion-associated HBV DNA from M198P mutant and the envelope rescue mutants were analyzed by qPCR as described in Fig. 8 63 Fig. 15 Similar levels of HBV RNAs were detected between wild type and small loop mutants in HuH-7 cells by Northern blot analysis 64 Fig. 16 Similar levels of HBV RNAs were detected between wild type and small loop mutants in HepG2-NTCP cells by Northern blot analysis 65 | |
| dc.language.iso | en | |
| dc.subject | 脯胺酸置換 | zh_TW |
| dc.subject | 封套膜蛋白 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | 小環狀結構 | zh_TW |
| dc.subject | 病毒顆粒分泌 | zh_TW |
| dc.subject | Proline substitution | en |
| dc.subject | Hepatitis B virus | en |
| dc.subject | HBV | en |
| dc.subject | Virion secretion | en |
| dc.subject | Envelope protein | en |
| dc.subject | Small loop | en |
| dc.title | 探討B型肝炎病毒小型封套膜蛋白之小環狀結構對於病毒顆粒型態發生及分泌所扮演的角色 | zh_TW |
| dc.title | The role of the small loop of hepatitis B virus small envelope protein in virion morphogenesis and secretion | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.author-orcid | 0000-0003-2639-9649 | |
| dc.contributor.oralexamcommittee | 吳妍華(Ho-Lin Chen),余明隆(Tian-Li Yu),葉昭廷,楊宏志,劉旻禕 | |
| dc.subject.keyword | B型肝炎病毒,病毒顆粒分泌,封套膜蛋白,小環狀結構,脯胺酸置換, | zh_TW |
| dc.subject.keyword | Hepatitis B virus,HBV,Virion secretion,Envelope protein,Small loop,Proline substitution, | en |
| dc.relation.page | 65 | |
| dc.identifier.doi | 10.6342/NTU202200130 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-01-22 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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