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Title: | 染色質重塑蛋白CHD4及RBBP4在EB病毒溶裂期的調控 The roles of chromatin remodelers CHD4 and RBBP4 in EBV lytic cycle |
Authors: | Yen-Chia Lin 林彥嘉 |
Advisor: | 陳美如 |
Keyword: | EB病毒,BMRF1聚合?輔助因子,染色質重塑複合體,CHD4,RBBP4, EBV,BMRF1,chromatin remodeling complex,CHD4,RBBP4, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | EB病毒屬於人類第四型皰疹病毒,EB病毒感染會造成感染性單核球增多症,此外感染EB病毒也與多種惡性腫瘤具有高度相關性。EB病毒的生活史可以分為潛伏期及溶裂期,此二階段分別具有不同的基因表現。在溶裂期複製階段,EB病毒聚合酶輔助因子BMRF1參與了多種功能的調控,除了能夠使聚合酶BALF5穩定結合於染色質以促進病毒複製,BMRF1也參與調控多個病毒溶裂期早期及晚期基因的轉錄作用。我們致力於探討BMRF1對於病毒溶裂期基因轉錄作用的調控機制。在實驗室先前的研究當中,經由免疫沉澱法結合質譜分析,染色質重塑複合體NuRD的成員CHD4及RBBP4被發現會與BMRF1之間具有交互作用。為了進一步探討CHD4及RBBP4在EB病毒溶裂期所扮演的角色,我們在EB病毒潛伏感染的鼻咽癌上皮細胞株NA中,首先利用免疫螢光染色以及免疫沉澱法確認在病毒被活化時,BMRF1會透過轉活化區段與CHD4及RBBP4產生交互作用。利用shRNA降低CHD4基因表現,會使病毒複製效率、溶裂期蛋白質表現量以及病毒顆粒分泌量下降,然而以shRNA降低RBBP4基因表現卻使得細胞內EB病毒基因量以及病毒顆粒分泌數量上升。利用螢光素酶報導基因檢測試驗,顯示CHD4能夠促使BMRF1所調控在oriLyt附近的BHLF1啟動子的活性上升。除此之外, RBBP4 會與BMRF1以及BrdU所標記新合成的DNA共同聚集在細胞核當中,顯示RBBP4可能參與協助病毒DNA的複製過程。綜合以上實驗結果,我們發現CHD4和RBBP4雖然同為NuRD complex成員,但可能分別透過不同的方式調控EB病毒溶裂期的基因表現,CHD4在病毒溶裂期中扮演了支持DNA複製的角色,RBBP4的角色與功能則需要進一步探討。 Epstein-Barr virus (EBV) is a gamma-herpesvirus that causes infectious mononucleosis and is highly associated with various malignancies. The life cycle of EBV are divided into latent and lytic stages with different gene expression profiles. Upon lytic reactivation, BMRF1, the viral DNA polymerase processivity factor, stabilizes the DNA binding ability of DNA polymerase BALF5 to facilitate EBV DNA replication. In addition, BMRF1 functions as a transcriptional regulator to activate the expression of several viral early and late genes. We are interested in exploring possible functions of BMRF1 in gene regulation. In our previous study, CHD4 and RBBP4, the components of the chromatin remodeling complex NuRD, were identified as BMRF1-interacting proteins by IP-mass spectrometry analysis. The interactions between BMRF1-CHD4 and BMRF1-RBBP4 during EBV lytic replication were confirmed by immunofluorescence assay and co-immunoprecipitation assay. Furthermore, we demonstrated that the transactivation domain of BMRF1 is responsible CHD4 and RBBP4 interactions. Knockdown of CHD4 reduced the viral DNA replication efficiency, lytic protein expression, and virion secretion in EBV-reactivated NA cells; whereas knockdown of RBBP4 showed increase in intracellular EBV genome copy number and virion secretion. In addition, the luciferase reporter assay revealed that CHD4 may facilitate BMRF1-mediated activation of BHLF1 promoter. Moreover, the BrdU incorporation assay suggested that RBBP4, but not CHD4, co-localized with viral lytic DNA replication compartment. Taken together, the results suggest that CHD4 and RBBP4 may function independently of the NuRD complex. CHD4 functions as a supportive role in EBV lytic cycle, but the role of RBBP4 in EBV lytic cycle needs further investigation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74930 |
DOI: | 10.6342/NTU201902425 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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