探討 BFRF1 與ESCRT 模組在EB 病毒出核時所扮演 的角色與機制

Abstract

當EB 病毒進入宿主細胞後，其線型DNA 會進到細胞核內成為環型結構，當病毒於細胞內呈潛伏期時，其DNA 會隨著細胞週期進行複製。當病毒進入溶裂期複製，病毒DNA 在核內完成複製與殼體組裝後成為核殼體，會藉由出核複合體BFRF1/BFLF2 促進核殼體由細胞核穿越核膜並釋出至細胞質。實驗室先前證明BFRF1 會透過Alix 吸引 ESCRT (endosomal sorting complex required for transport) 膜剪切模組幫助BFRF1 產生核模衍生液泡 (nuclear envelope-derived vesicle) 以加速核殼體出核。由於ESCRT 系統是由三十多個成員串連而執行功能，已知參與細胞內膜的剪接運輸及多種病毒的成熟過程。對於ESCRT 如何參與核膜結構的改變仍只有少數研究報導，因此在本論文中首先探討BFRF1 如何利用其不同功能區域吸引Alix 並將核膜塑形產生核膜衍生液泡。同時利用不同ESCRT 成員的shRNA 使其表現量下降，以探討那些ESCRT 膜剪切蛋白成員會參與在EB 病毒核殼體出核的過程。為了之後得 到大量的BFRF1 蛋白質以建構抗體，本論文也嘗試建立大腸菌產生的重組BFRF1 蛋白質表現系統。本論文之結果發現(I)在共免疫沉澱試驗中發現BFRF1 會分別利用LD1 (8-65 a.a) 與ESR (180-313 a.a) 和Alix 的Bro (1-358 a.a)及PRR (703–868 a.a) 功能區域結合。當 BFRF1 這兩個區域的刪除或是點突變會影響到細胞內Alix 的分布及 BFRF1 產生核膜衍生液泡的能力。(II)同時也利用含有EB 病毒的鼻咽癌上皮細胞NA 觀察，以shRNA 病毒使ESCRT 表現量下降時，會影響釋出細胞的病毒數量。(III)利用大腸桿菌表現大量重組BFRF1 蛋白，可用於生產高專一性的抗體，以探討BFRF1 在 EB 病毒核殼體出核過程的路徑與機制。這些發現將有助於了解BFRF1 在調控核膜塑形上的分子機制。

Upon its entrance into the cell, the linear EBV genome is injected into the nucleusand becomes circular episomes, which can be maintained in these cells during latent infection. When virus is induced into lytic replication, the replicated viral genomes are packaged into procapsids to form nucleocapsids in the nucleus before translocated into the cytoplasm for subsequent maturation process. The nuclear egress complex (NEC) composed of BFRF1 and BFLF2 can facilitate the budding of nucleocapsids across the nuclear envelope (NE). Previously, our laboratory emonstrated that BFRF1 interacts with Alix to recruit ESCRT (endosomal sorting complex required for transport) machinery to induce nuclear envelope-derived vesicles for promoting nuclear egress. ESCRT machinery is composed of at least 30 proteins to mediate membrane scission and cytoplasmic budding of various virus. How the ESCRT system is involved in modulating nuclear envelope structure is just immerging. Therefore, in the first part of this study, we intend to define how the viral membrane protein BFRF1 recruits Alix and the functional domains required for nuclear envelope-derived vesicle formation. Secondly, a shRNA approach is used to knockdown individual ESCRT components to examine the participation of different ESCRT components in EBV nuclear egress or mature virion secretion. Bacterially expressed recombinant BFRF1 protein expression system was used to obtain purified BFRF1 for generating antibody. Three different aspects of BFRF1 is achieved in this study. (I) Co-immunoprecipitation results showed that LD1 (8-65 a.a ) and ESR (180- 313 a.a) domains of BFRF1 interacted with Alix Bro (1-358 a.a) and PRR (703-868 a.a), respectively. Site-directed mutagenesis of LD1 or ESR in BFRF1 reduced BFRF1 induced vesicle formation and affected cellular Alix subcellular distribution. (II) shRNA targeting various ESCRT components were used in EBV positive epithelial cells NA, the preliminary data indicate that knockdown of ESCRT components does affect virion release but not viral DNA replication. (III)A bacterially recombinant BFRF1 protein was expressed and purified for generating highly specific antibody to study the intracellular trafficking of BFRF1 during EBV nuclear egress process. Overall, this study will help to reveal the molecular mechanisms of BFRF1-mediated budding and scission of EBV nuclear egress vesicles.