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標題: | 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 Study of HIV-1 CRF07_BC particle assembly and budding |
作者: | Chun-Kai Chang 張浚凱 |
指導教授: | 張淑媛(Sui-Yuan Chang) |
關鍵字: | CRF07_BC亞型病毒,Gag前驅蛋白,ESCRT,p6, CRF07_BC,Gag,ESCRT,p6, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57706 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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