EB 病毒 BFRF1 引發核膜衍生液泡的機制及其對物質由細胞核到質運送之調控

The mechanism of EBV BFRF1 induced nuclear envelope-associated vesicle formation and its regulatory role on nucleocytoplasmic transport

劉冠婷(Guan-Ting Liu)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10124

摘要

ESCRT (Endosomal Sorting Complexes Required for Transport) 機制已知會參與在細胞中膜剪切的過程，以及許多病毒在細胞質膜出芽離開細胞的過程。在先前的研究中，我們發現EB病毒會藉由內核膜蛋白BFRF1與ESCRT的橋接分子Alix作用，幫助核膜衍生液泡結構的形成，可助於已包裹完成的病毒衣殼 (nucleocapsid) 離開細胞核。在本篇研究中，進一步探討BFRF1協同ESCRT蛋白調控核膜結構產生液泡的分子機制。透過時程分析蛋白分佈，我們發現BFRF1會先後吸引不同的ESCRT分子到細胞核膜，其中 Alix 與 TSG101會先後靠近核，進而幫助液泡結構的形成。更進一步利用結構域突變株研究BFRF1與Alix結合區，透過共同免疫沉澱法、麩胺基硫轉移酶沉降及螢光染色訊號，發現在BFRF1胺基酸61-69之中，存在著一段非典型的Alix結合區。BFRF1上面的這一段Alix結合區主要會與Alix的Bro domain有交互作用，且處理核酸酶之後，不會影響BFRF1與Alix的結合。進一步，我們也探討BFRF1造成的液泡結構幫助細胞核巨分子運送的可能性。當細胞表現BFRF1時可將細胞核中不正常累積的錯誤摺疊蛋白運送到細胞質中，進而藉由自噬作用將這些錯誤摺疊蛋白進行降解。暗示著細胞中原本可能存在一條路徑，可以調控細胞核與細胞質之間巨分子的運輸及不正常堆積蛋白的降解。這篇研究提供了EB病毒BFRF1引發核膜衍生液泡的機制及其對物質由細胞核到質運送之調控。

關鍵字

EB病毒 ； BFRF1 ； Alix ； TSG101 ； 液泡結構形成 ； 自噬 ； 細胞核與細胞質間的運輸

並列摘要

The cellular endosomal sorting complex required for transport (ESCRT) machinery mediates membrane scission and cytoplasmic budding of various viruses. Previously, we showed that Epstein-Bar virus (EBV) BFRF1 recruits ESCRT component Alix to the nuclear envelope-associated membrane for virus maturation. In this study, the mechanism used by BFRF1 for modulating nuclear envelope was further characterized. ESCRT components were recruited sequentially to the nuclear envelope along with BFRF1 expression by time-course analysis. Alix was recruited at the beginning of nuclear envelope modulation, whereas the other ESCRT component TSG101 was recruited to nuclear rim at late stages for cytoplasmic vesicle release. By serial deletion and site-directed mutagenesis, a nonconventional late domain on BFRF1 was identified to mediate the Bro and PRR domains of Alix recruitment. Remarkably, depletion of nuclear acid did not abolish the associations between BFRF1 and Bro domain, suggesting BFRF1 is directly interact with Aix Bro domains. Investigating the possible capability of BFRF1-mediated vesicles for delivering nuclear component, we found that expression of BFRF1 reduced the nuclear accumulation of protein aggregates. The reduced nuclear aggregates was recovered or enhanced by autophagy inhibitor or inducer, suggesting cytoplasmic autophagy system is involved in the nuclear envelope-directed molecule transport. Overall, this study provides understandings not only for virus mediated nuclear envelope modulation, but also the mechanism of nucleocytoplasmic transport.

並列關鍵字

EBV ； BFRF1 ； Alix ； TSG101 ； vesicle formation ； autophagy ； nulear aggregates ； nucleocytoplasmic transport

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