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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張淑媛(Sui-Yuan Chang) | |
dc.contributor.author | Chun-Kai Chang | en |
dc.contributor.author | 張浚凱 | zh_TW |
dc.date.accessioned | 2021-06-16T06:59:05Z | - |
dc.date.available | 2015-10-09 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-17 | |
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Yeager, Atomic-level modelling of the HIV capsid. Nature, 2011. 469(7330): p. 424-7. 68. Meng, X., et al., Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid. PLoS Pathog, 2012. 8(8): p. e1002886. 69. Llewellyn, G.N., et al., Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells. PLoS Pathog, 2010. 6(10): p. e1001167. 70. Campbell, S. and A. Rein, In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain. J Virol, 1999. 73(3): p. 2270-9. 71. Sandefur, S., et al., Mapping and characterization of the N-terminal I domain of human immunodeficiency virus type 1 Pr55(Gag). J Virol, 2000. 74(16): p. 7238-49. 72. Wright, E.R., et al., Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J, 2007. 26(8): p. 2218-26. 73. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57706 | - |
dc.description.abstract | HIV-1 CRF07_BC重組亞型病毒於2004至2006年間在台灣的靜脈毒癮者間爆發感染,臨床觀察得知CRF07_BC病毒感染造成之疾病進程較其它HIV-1亞型緩慢。先前本實驗室研究發現CRF07_BC的p6上有7個胺基酸刪除,且刪除位置恰巧位於gag/pol閱讀框架之重疊處。由於此7個胺基酸刪除位於p6Pol病毒蛋白酶第三切割點的上游,因此會干擾病毒蛋白酶的活化,進而降低CRF07_BC病毒顆粒成熟過程中Gag前驅蛋白的切割效率,導致較多不成熟且感染力較差的病毒產生。同時也發現CRF07_BC病毒的相對釋放係數較低及病毒顆粒栓繫於細胞膜上的現象,顯示其病毒顆粒釋出有減少的趨勢。
為探討造成CRF07_BC病毒釋出減少的原因,我們對其病毒顆粒組裝與釋出過程中的各個步驟進行檢視。由於Gag前驅蛋白在HIV-1病毒顆粒的組裝與釋出扮演重要角色,我們首先確認CRF07_BC病毒Gag前驅蛋白並不會在細胞內受到病毒蛋白酶的提早切割,顯示其Gag前驅蛋白產生後應可順利進入後續組裝釋出的步驟。接著,因為Gag前驅蛋白會利用p6功能區域結合宿主細胞中的ESCRT複合體進而協助病毒顆粒自細胞膜釋出,因此我們以免疫共沉澱實驗及共軛焦免疫螢光染色證明CRF07_BC病毒Gag前驅蛋白與ESCRT複合體中的Tsg101及Alix蛋白之間皆存在一定之交互作用,顯示CRF07_BC病毒在利用ESCRT複合體協助病毒顆粒釋出的特性並無發生缺陷。最後,Gag前驅蛋白在細胞內的互相結合與聚集為HIV-1病毒顆粒組裝之關鍵步驟,我們以共軛焦免疫螢光染色觀察CRF07_BC病毒Gag前驅蛋白於細胞內的交互作用,發現CRF07_BC病毒Gag前驅蛋白於細胞內的聚集程度較B亞型病毒低,此一現象在將CRF07_BC病毒p6上缺失的7個胺基酸補回後可獲得補償,使Gag前驅蛋白恢復交互作用。 | zh_TW |
dc.description.abstract | In 2004–2006, an outbreak of HIV-1 CRF07_BC infections was observed among intravenous drug users (IDUs) in Taiwan, and patients infected with CRF07_BC exhibited a slower immunological progression. A 7-amino acid deletion in the p6 region of CRF07_BC was observed. The 7-amino acid deletion is loacated immediately upstream of the 3rd protease cleavage site and is reported to cause defective protease activation and reduced Gag polyprotein processing, which subsequently resulted in production of immature and non-infectious viral particles. In addition, decreased virus release and tethering of viral particles on the plasma membrane was observed, suggesting a reduced efficiency of CRF07_BC virus release.
The involvement of Gag in virus assembly and budding was investigated. First, the reduced processing of CRF07_BC Gag is not due to early processing based on the time-course experiment. Second, the 7-amino acid deletion in p6 has neglectable effect on its interaction with both Tsg101 and Alix, members in endosomal sorting complex required for transport (ESCRT), by co-immunoprecipitation and confocal immunofluorescence assays. Third, a significant reduction of CRF07_BC Gag-Gag interaction and multimerization in cytoplasm was observed by confocal immunofluorescence, and complementation of the 7-amino acid in the CRF07_BC p6 could rescue this defective phenotype. In this study, we demonstrated that the 7-amino acid deletion in p6 could decrease Gag-Gag interaction, which together with reduced activation of protease, contribute to the inefficient CRF07_BC release. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T06:59:05Z (GMT). No. of bitstreams: 1 ntu-103-R01424009-1.pdf: 12672224 bytes, checksum: 79fde92779f3d5f1f49c115dbf2aadac (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 III 英文摘要 IV 圖目錄 IX 表目錄 X 第一章 緒論 1 1-1. 人類免疫缺陷病毒(HIV)與後天免疫缺乏症候群(AIDS) 1 1-2. HIV-1之流行現況 1 1-3. HIV-1病毒構造簡介 2 1-4. HIV-1病毒組裝(Assembly)及釋出(Budding) 3 1-4.1. 病毒組裝(Assembly)與Gag前驅蛋白(Pr55Gag) 3 1-4.2. 病毒釋出(Budding)與內吞體分選轉運複合體(ESCRT) 4 1-4.3. 抗病毒限制因子(Anti-viral restriction factors) 6 1-5. HIV-1 CRF07_BC重組亞型病毒之功能特性 7 1-5.1. CRF07_BC感染之疾病進程 7 1-5.2. CRF07_BC之病毒基因與表型(Phenotype) 7 1-6. 研究動機 9 第二章 實驗材料與方法 10 2-1. 實驗材料 10 2-1.1. 細胞 10 2-1.2. 商業試劑套組 10 2-1.3. 抗體 10 2-1.4. 質體 11 2-2. 實驗方法 12 2-2.1. 293T細胞株培養 12 2-2.2. 轉染作用 (Transfection) 12 2-2.3. 蛋白質電泳 (SDS-polyacrylamide gel electrophoresis, SDS-PAGE) 13 2-2.4. 西方墨點法 (Western blot) 13 2-2.5. 免疫共沉澱法 (Co-immunoprecipitation assay, Co-IP) 14 2-2.6. 間接免疫螢光染色 (Indirect immunofluorescence, IF) 14 2-2.7. 細胞RNA萃取 (RNA extraction) 15 2-2.8. RNA反轉錄實驗 (Reverse transcription) 15 2-2.9. HIV-1 B亞型病毒HXB2之Gag基因聚合酶連鎖反應 15 2-2.10. HIV-1 CRF07_BC亞型病毒之Gag基因聚合酶連鎖反應 16 2-2.11. Alix基因聚合酶連鎖反應 16 2-2.12. Tsg101基因聚合酶連鎖反應 17 2-2.13. Tetherin基因聚合酶連鎖反應 17 2-2.14. pCRTMII-TOPOR連接(Ligation)反應 18 2-2.15. 洋菜膠中萃取DNA片段(DNA gel purification) 18 2-2.16. T4 DNA連接酶反應(T4 DNA ligase ligation) 19 2-2.17. 轉型作用(Transformation) 19 2-2.18. 小量細菌質體萃取(Mini plasmid extraction) 19 2-2.19. 大量細菌質體萃取(Maxi plasmid extraction) 20 第三章 實驗結果 22 3-1. CRF07_BC亞型病毒Vpu與Tetherin之交互作用 22 3-2. CRF07_BC亞型病毒Gag前驅蛋白未受提前切割 22 3-3. 建構質體表現Gag前驅蛋白、Tsg101及Alix 23 3-3.1. 建構pcDNA3.1-Gag質體表現Gag前驅蛋白 24 3-3.2. 建構pRK5 -Tsg101質體表現Tsg101蛋白 25 3-3.3. 建構pRK5-Alix質體表現Alix蛋白 25 3-4. CRF07_BC亞型病毒Gag前驅蛋白與ESCRT之交互作用 26 3-4.1. CRF07_BC亞型病毒Gag前驅蛋白與Tsg101之交互作用 26 3-4.2. CRF07_BC亞型病毒Gag前驅蛋白與Alix之交互作用 28 3-4.3. HIV-1 質體表現 Gag前驅蛋白與Alix蛋白之交互作用 28 3-5. CRF07_BC亞型病毒Gag前驅蛋白之間的交互作用 29 3-5.1. pcDNA3.1-Gag表現Gag前驅蛋白之交互作用 30 3-5.2. HIV-1 molecular clone表現Gag前驅蛋白之交互作用 31 第四章 實驗討論 33 第五章 參考文獻 38 | |
dc.language.iso | zh-TW | |
dc.title | 探討人類免疫缺乏病毒CRF07_BC重組亞型病毒株之病毒顆粒組裝與釋出 | zh_TW |
dc.title | Study of HIV-1 CRF07_BC particle assembly and budding | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 高全良(Chuan-Liang Kao),李君男(Chun-Nan Lee),劉旻禕(Min-Yi Liu) | |
dc.subject.keyword | CRF07_BC亞型病毒,Gag前驅蛋白,ESCRT,p6, | zh_TW |
dc.subject.keyword | CRF07_BC,Gag,ESCRT,p6, | en |
dc.relation.page | 72 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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