EB病毒BGLF4蛋白質激酶進核機制及對物質進核調控之探討

The nuclear targeting of EBV BGLF4 kinase and its regulatory effects on nuclear import

張洲維(Chou-Wei Chang)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10501

摘要

BGLF4是EB病毒（Epstein-Barr virus）中唯一的Ser/Thr蛋白質激酶，它會磷酸化許多病毒本身或宿主細胞的受質使得周圍環境適合病毒複製和病毒核殼體（nucleocapsid）的出核。在病毒複製的早期及晚期，BGLF4蛋白質表現主要分佈在細胞核中，但是有少部份的BGLF4會在複製晚期進入細胞質並被包裹於病毒顆粒裡。因此，本研究分成兩個主題來探討。第一是找出與BGLF4蛋白質進核（nuclear import）有關的功能性區域和機制。首先藉由系統性分析蛋白質功能性區域，發現BGLF4的N端和C端都扮演重要的調控角色。進一步透過共免疫沈澱法和蛋白質體外結合實驗（in vitro GST pull down）發現BGLF4會和核孔蛋白（nucleoporins, Nup）Nup62和Nup153有交互作用。尤其是利用HeLa細胞透化作用（permeabilize）進行進核實驗更證明了BGLF4進核過程是不須要細胞質中的調控分子協助。由於大多數的物質進入細胞核皆須透過核定位訊號（nuclear localization signal）協同細胞質調控分子與核孔蛋白交互作用，因此本研究證明BGLF4是透過一個新的進核機制達到細胞核中。在第二部份則進一步去探討BGLF4蛋白質對核孔複合體（nuclear pore complex, NPC）的結構影響及進核功能的調控。透過共軛焦顯微鏡分析，發現BGLF4蛋白質激酶會利用其激酶活性去影響Nup62和Nup153在細胞中的分佈。進一步實驗中發現，BGLF4會去抑制進核蛋白beta（importin beta）的進核，使得需要透過典型核定位訊號進核的蛋白質無法進入細胞核。值得注意的是BGLF4可以幫助不具有核定位訊號的病毒蛋白進核，包括病毒的複製蛋白BSLF1、BBLF2/3、BBLF4和主要的殼體蛋白VCA。綜合以上實驗結果，本研究發現BGLF4蛋白質激酶會透過干擾Nup62和Nup153的正常功能而幫助病毒蛋白的進核，進而促進病毒的DNA複製和殼體的組裝（assembly）。

關鍵字

EB病毒 ； BGLF4 ； 核孔複合體 ； 核孔蛋白 ； 進核

並列摘要

BGLF4 of Epstein-Barr virus (EBV) encodes a serine/threonine protein kinase that phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and nuclear egress of viral nucleocapsids. BGLF4 is expressed predominantly in the nucleus at early and late stages of virus replication, while a small portion of BGLF4 is distributed in cytoplasm at late stage of virus replication and packaged into the virion. In this study, two specific aims are addressed. The first part is to identify the functional domains and the mechanism involved in the nuclear localization of BGLF4. We analyzed the functional domains crucial for nuclear localization of BGLF4 and found both N- and C-termini play important modulating roles. Both co-immunoprecipitation and in vitro pull-down assays further demonstrated that BGLF4 binds to Nup62 and Nup153. Remarkably, nuclear import assay with permeabilized HeLa cells demonstrated that BGLF4 translocated into nucleus independent of cytosolic factors. Data here suggest BGLF4 employs a novel mechanism through direct interactions with nucleoporins for its nuclear targeting. The second part is to investigate the regulatory effects of BGLF4 on the structure and biological functions of nuclear pore complex (NPC). We found that BGLF4 modified the distribution pattern of Nup62 and Nup153 in a kinase activity-dependent manner in confocal microscopy analysis. Further, the nuclear targeting of importin-beta was attenuated in the presence of BGLF4, leading to the inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. Notably, we found BGLF4 promoted the nuclear import of several non-NLS containing EBV proteins including viral DNA replicating enzymes BSLF1, BBLF2/3 and BBLF4 and the major capsid protein (VCA). Taken together, BGLF4 interferes with the normal functions of Nup62 and Nup153, and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly.

並列關鍵字

EBV ； BGLF4 ； nuclear pore complex ； nucleoporins ； nuclear import

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