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標題: | Epstein-Barr virus BBRF2蛋白質的功能性分析 Functional analysis of Epstein-Barr virus BBRF2 protein |
作者: | Hsiang-Hung Huang 黃翔弘 |
指導教授: | 張麗冠 |
關鍵字: | EBV,間質蛋白質,晚期基因,自噬作用, EBV,tegument proteins,late gene,autophagy, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | Epstein-Barr virus (EBV)屬於人類皰疹病毒,具有感染上皮細胞及B淋巴細胞的能力,並且是第一個被發現的人類致癌病毒。EBV生活史可分為潛伏期以及溶裂循環,而EBV為了在溶裂循環完成病毒顆粒的複製、組裝及運送會表現出極早期、早期及晚期基因,但目前為止仍有數種蛋白質的功能仍未被仔細研究。然而在EBV中的BBRF2蛋白質,其同源物 (homolog)普遍存在於其他皰疹病毒中,且功能大多與病毒顆粒的晚期功能如組裝與釋放有關,但目前的研究尚未發現EBV的BBRF2蛋白質在其生活史中的功能性。因此本研究首先建立BBRF2的表現系統,純化E. coli中的His-tagged BBRF2以製備抗體,藉此分析其蛋白質特性。然後利用含有EBV的P3HR1細胞以TPA/sodium butyrate誘導病毒引發溶裂循環,以RT-PCR分析BBRF2 mRNA的表現,結果在12小時開始表現,並於之後漸多,約在進入溶裂循環60小時後達到高峰,顯示BBRF2是一種溶裂期的基因。另外透過共軛焦螢光顯微鏡觀察BBRF2在EBV進入溶裂循環後,蛋白質在細胞內的分佈情形,結果顯示,在溶裂循環引發48小時後,BBRF2會與病毒極早期基因Rta在細胞核外有部分重合的現象,在72小時後則會與病毒晚期蛋白質 VCA (Major capsid protein)在細胞核外有部分重合的現象。由於許多病毒的晚期功能被證明與細胞內的各種功能性胞器有密切相關,因此本研究利用融合蛋白GFP-LC3標定細胞內的自噬體 (autophagosome),在48小時透過共軛焦螢光顯微鏡觀察到BBRF2與LC3在細胞核外具有重合的現象,因此推測BBRF2與自噬體的形成可能有關。另外也進一步觀察到選擇性自噬作用 (selective autophagy)中的cargo-protein p62,在細胞自噬作用被誘導後會與BBRF2在細胞質有重合的現象。而在觀察誘導進入溶裂循環的P3HR1細胞中LC3-I轉變成 LC3-II的情形中,發現在EBV的溶裂循環時,LC3-II會增加,表示其自噬作用被誘導。總結以上,BBRF2對EBV在引發溶裂循環後可能扮演重要的角色,未來將更深入探討BBRF2與細胞自噬作用之間的關係。 Epstein-Barr virus (EBV) is a human herpesvirus, which infects epithelial cells and B lymphocytes. EBV is the first discovered human carcinoma virus. The life cycle of EBV can be divided into latency and lytic cycle. During the lytic cycle, EBV expresses the immediate-early, early and late genes in order to complete viral replication, viral particles assembly and viral egress. So far, some of EBV proteins’ functions remain unknown. BBRF2 is conserved in all other herpesvirus family, and its in other herpesvirus have been found to involved in virus particle assembly and release. However, the functions of BBRF2 in EBV remain be characterized. In this study, anti-BBRF2 antibody was generated to analyze BBRF2 functions using E. coli expressed His-tagged BBRF2. RT-PCR analysis revealed that the expression of BBRF2 mRNA occurs at 12 hr post induction by TPA/sodium butyrate in P3HR1 cells. The maximum amount of BBRF2 mRNA is at 60 hr after lytic induction, suggesting that BBRF2 is a lytic gene. Moreover, BBRF2 colocalizes with Rta in the cytoplasm as dots at 48 hr after lytic induction by confocal microscopy. At 72 hr post induction, BBRF2 also colocalizes with major capsid protein (VCA) in the cytoplasm. During the late stage of EBV life cycle, some viral proteins interact with different cell organelles in order to complete its life cycle. By using GFP-LC3 as an autophagosome marker, BBRF2 and Rta colocalize with LC3 in the cytoplasm after 48 hours induction, revealing that BBRF2 is related to autophagosome formation. Furthermore, BBRF2 colocalizes with p62, a cargo protein in selective autophagy, in the cytoplasm after starvation. Finally, this study found LC3-I is converted into LC3-II during lytic induction, suggesting that lytic cycle induces autophagy. Taken together, BBRF2 plays a crucial role in EBV lytic activation. The relationship between BBRF2 and autophagy will be elucidated in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64514 |
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顯示於系所單位: | 生化科技學系 |
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