BMRF1調節EB病毒溶裂期基因表現及影響BKRF3 DNA-尿嘧碇糖苷酶功能之探討

BMRF1-Mediated Regulation of Viral Gene Expression and the Biological Function of BKRF3 Uracil-DNA Glycosylase in Epstein-Barr Virus Lytic Replication

蘇美慈(Mei-Tzu Su)

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(2015年)

https://doi.org/10.6342/NTU.2015.11208

摘要

EB病毒是普遍感染全球人口的gamma型疱疹病毒。EB病毒與Burkitt’s淋巴瘤、Hodgkin’s症、鼻咽癌等許多人類惡性疾病形成有高度相關性。EB病毒感染後，環狀病毒基因體利用細胞內DNA複製機組進行複製並潛伏在宿主細胞中。 EB病毒由潛伏再活化後，轉活化因子Zta及Rta的表現會活化一連串溶裂期基因表現以利病毒複製。EB病毒與哺乳細胞之DNA複製複合體類似，包含導引酶(primase, BSLF1)、導引酶輔助蛋白(primase accessory protein, BBLF2/BBLF3)、解螺旋酶(helicase, BBLF4)、DNA聚合酶(DNA polymerase, BALF5)、單股DNA結合蛋白質(single-stranded DNA-binding protein, BALF2)、尿嘧啶醣苷酶(uracil-DNA glycosylase, BKRF3)和DNA聚合酶輔助因子(DNA polymerase processivity factor, BMRF1)已被證實會聚集在病毒DNA複製區。BMRF1除了幫助病毒DNA複製，同時也可作為轉活化因子調節溶裂期病毒基因體複製起始區的BHLF1啟動子，或作為共同活化因子調節單股DNA結合蛋白BALF2啟動子，顯示BMRF1在病毒溶裂期DNA複製及基因表現都扮演重要角色。本論文中對於BMRF1相關生物功能進行兩個研究主題: (I) BMRF1調控BKRF3進入細胞核，以及BKRF3在溶裂期病毒核酸複製複合體中所扮演的生物功能。研究結果發現BMRF1可與BKRF3交互作用進而促使BKRF3由細胞質進入細胞核中，而BKRF3與DNA複製相關蛋白的結合可以提高BKRF3之酵素活性。剔除BKRF3基因導致病毒DNA複製功能受損, 並且以互補試驗證明BKRF3的leucine loop對BKRF3協助病毒基因體複製是重要的。(II)利用基因剃除病毒系統探討BMRF1對病毒基因的轉錄調控。實驗結果發現BMRF1參與部分病毒晚期基因之轉錄調控，而BMRF1轉活化功能區會影響其調控病毒基因表現的功能。以免疫沉澱法搭配質譜科學分析法(Immunoprecipitation-mass spectrometry)分析與BMRF1相互作用之蛋白質，發現BMRF1不僅與DNA複製/修復及RNA剪接相關蛋白形成蛋白質複合體，也與染色質調控分子像是BRG1 (BRM/SWI2-related gene 1)有相互作用。進一步發現BMRF1與BRG1在細胞內及體外試驗皆有交互作用。以干擾RNA抑制BRG1表現會降低BMRF1對部分病毒啟動子的轉活化功能，顯示BRG1可能參與BMRF1調節的病毒基因之轉錄調控。本論文證明BMRF1調控BKRF3參與細胞核內複製複合體是病毒DNA複製的關鍵；除了參與病毒基因體複製，BMRF1也可能與染色質調控分子BRG1共同調控病毒基因表現以利EB病毒溶裂期複製。

關鍵字

EB病毒 ； DNA聚合?輔助因子BMRF1 ； 尿嘧啶醣??BKRF3 ； 病毒核酸複製 ； 病毒基因表現

並列摘要

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects most of the population worldwide. EBV is highly associated with various human malignancies, including Burkitt’s lymphoma (BL), Hodgkin’s disease (HD) and nasopharyngeal carcinoma (NPC). After primary infection, the circularized EBV genome persists latently in the host cell, using cellular DNA replication machinery for replication. Once EBV reactivation from latency, the expression of transactivators Zta and Rta turn on cascade expression of viral lytic genes which are required for efficient viral replication. Similar to mammalian DNA replication machinery, a large protein complex including primase (BSLF1), primase accessory protein (BBLF2/3), helicase (BBLF4), DNA polymerase (BALF5), single-stranded DNA-binding protein (BALF2), uracil-DNA glycosylase (UDG, BKRF3), and DNA polymerase processivity factor (BMRF1) are demonstrated to associate with viral DNA replication compartment. Other than the role in DNA replication, BMRF1 also functions as a transactivator on BHLF1 promoter and as a coactivator on BALF2 promoter, indicating that BMRF1 plays important roles in viral DNA replication and viral gene expression. To explore the biological functions of BMRF1, two specific aims are addressed in this study. (I) The first part is to explore whether BMRF1 mediates nuclear translocation of BKRF3 and to study the biological function of BKRF3 in viral DNA replication complex during lytic cycle. It was found that BMRF1 interacts with BKRF3 and regulates the nuclear targeting of BKRF3. The interaction of BKRF3 with viral proteins in replication compartment enhances the enzymatic activity of BKRF3 during lytic cycle. In BKRF3 knockout recombinant virus, lytic DNA replication is blocked and can be rescued by trans-complementation of BKRF3, indicating that BKRF3 is required for EBV DNA replication and the leucine loop of BKRF3 is critical for its function. (II) The second part is to investigate the transcription regulatory function of BMRF1 using specific gene knockout bacmid system. In gene regulation analyses, we found that BMRF1 regulates a number of viral late gene expression through its transactivation domain. According to immunoprecipitation-mass spectrometry analysis, BMRF1 associates with not only DNA replication/repair and RNA splicing association factors but also chromatin modifiers such as BRG1 (BRM/SWI2-related gene 1). Moreover, BMRF1 interacts with BRG1 in the cells and in vitro. Knockdown of BRG1 reduces transactivation activity of BMRF1 on a subset of viral promoters, suggesting BRG1 may participate in the regulation of BMRF1 on viral gene expression. In conclusion, BMRF1 recruits viral UDG BKRF3 to the replication compartment, which is crucial for viral DNA replication. In addition to the role in DNA replication machinery, BMRF1 may also mediate gene expression through interacting with chromatin modifier BRG1 to benefit EBV lytic replication.

並列關鍵字

Epstein-Barr virus ； DNA polymerase processivity factor BMRF1 ； uracil-DNA glycosylase BKRF3 ； viral DNA replication ； viral gene expression

參考文獻

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