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標題: | 探討EB病毒釋出過程中BFRF1蛋白之功能區域及ESCRT模組的參與 Characterization of the functional domains in EBV BFRF1 and the roles of cellular ESCRT components in virus maturation process |
作者: | 阮憶婷 Yi-Ting Juan |
指導教授: | 陳美如 |
關鍵字: | EB病毒,BFRF1,釋出過程,Alix,BFLF2,ESCRT-III, Epstein-Barr virus,BFRF1,virus release,Alix,BFLF2,ESCRT-III, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 當EB病毒進入溶裂期 (lytic replication) 後會開始在核內進行DNA複製,複製完成後的DNA會被包裹形成核殼體(nucelocapsid),核殼體會透過出核複合體(nuclear egress complex, NEC)的幫助離開細胞核進到細胞質。EB病毒的出核複合體是由BFRF1及BFLF2兩個病毒蛋白所組成,已知BFRF1會透過和細胞中的Alix進行交互作用進而吸引ESCRT (endosomal sorting complex required for transport)相關分子進行核膜剪切,幫助BFRF1產生核膜衍生液泡(nuclear envelope-derived vesicle)以加速病毒核殼體出核。本篇研究著重在探討病毒釋出過程中,BFRF1是透過哪些功能區域 (functional domains) 和Alix以及BFLF2進行交互作用,並透過初步篩檢找出哪些ESCRT-III分子可能參與在釋出過程中。之前實驗室研究發現BFRF1的Late domain 1 (LD1) 區域可以和Alix的Bro區域產生交互作用。本研究進一步發現BFRF1的EBV specific region (ESR) 區域可以和Alix的PRR區域產生交互作用,若將ESR區域上的正電荷進行點突變或是藉由核酸酶處理皆會影響兩者之間的交互作用,推測ESR和PRR之間的交互作用需要透過核酸的參與。另外在帶有EB病毒B95-8 strain bacmid (p2089)且剔除BFRF1的細胞 (293TetEZ/p2089BFRF1) 中,透過qPCR偵測發現病毒釋出可能會因為缺少這些和Alix進行交互作用的區域而下降。此外也發現BFRF1上的多段功能區域皆能和BFLF2產生交互作用,但刪除掉任何一段皆會影響BFRF1將BFLF2帶出核到核膜並形成共位的現象,顯示BFRF1可能需要依賴多段功能區域來執行其出核複合體的功能。而在BFLF2上觀察到胺基酸序列81-107為和BFRF1進行交互作用及共位的主要胺基酸序列。最後也篩選出可能參與在病毒釋出過程中的ESCRT-III分子,像是CHMP7以及CHMP2A。本研究除了闡明BFRF1的功能區域如何和Alix及BFLF2進行交互作用以利病毒核殼體出核,也篩選到在釋出過程中可能參與的ESCRT-III分子,以供未來之研究。 During lytic replication in the nucleus, Epstein-Barr virus genome is replicated and packaged into capsids. Subsequently, the assembled nucleocapsids egress from the nucleus through a nuclear envelope-derived vesicle dependent manner, which is mediated by the nuclear egress complex (NEC) composed of viral BFRF1 and BFLF2 proteins. Previously, we demonstrated that cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in the nuclear egress through the interaction between BFRF1 and the ESCRT-adaptor protein Alix. In this study, it is aimed to dissect the functional domains in BFRF1 required for the recruitment of Alix and BFLF2 and to identify the ESCRT-III components participated in the process. Previous study revealed that the Late Domain 1 (LD1) of BFRF1 interacts with Bro domain of Alix. Here, we further showed that EBV-specific region (ESR) interacts with PRR domain of Alix and the interaction is nucleic acid-dependent. Remarkably, virion secretion is diminished in BFRF1 knock-out 293TetEZ/p2089 EBV bacmid inducible cells after complemented with interacting domain-defective mutants. In addition, in the part of dissecting BFLF2-interacting domains, multiple regions of BFRF1 interact with BFLF2. All BFRF1 mutants with deletion of individual domain fail to recruit BFLF2 out of the nucleus, indicating that multiple regions of BFRF1 are required for its function. By contrast, a.a. 81-107 of BFLF2 is the major region responsible for the interaction with BFRF1. On the other hand, screening with various ESCRT-III dominant negative clones indicates that CHMP2A and CHMP7 may be required in the maturation process. Altogether, we characterize the functional domains of BFRF1 involved in the recruitment of Alix and BFLF2. The candidates of ESCRT-III components participated in the maturation process will be further accessed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79082 |
DOI: | 10.6342/NTU201802932 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-11 |
顯示於系所單位: | 微生物學科所 |
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