2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/651371摘要：EB 病毒(Epstein-Barr virus)是一種與許多人類惡性疾病有高度相關性的人類gamma疱疹病毒。EB 病毒最獨特之處就在於它具有重複從潛伏期活化進入溶裂期進行病毒基因體複製的能力，和一連串嚴密的基因調控機制。當病毒活化進入溶裂期時，包含有病毒複製必須啟動子(BHLF1)的溶裂期複製起點(oriLyt)區域可以和許多病毒與細胞轉錄活化因子相結合。目前已知許多的細胞訊息傳遞途徑皆可藉由影響這段複製起點區域來調控病毒從潛伏期進入溶裂期基因體複製，例如細胞中的NF-kappa B 活性被發現可透過阻斷病毒特早期活化子(immediate early transactivator)作用，進而抑制多種gamma 疱疹病毒的活化。除了病毒蛋白與細胞訊息傳遞的交互作用外，病毒的BGLF4 蛋白激酶也被發現可藉由調控許多病毒及細胞因子來促進病毒複製。BGLF4 是一種脯胺酸依賴性(proline-dependent) 蛋白激酶，具有cyclin-dependent kinase (CDK)活性的絲胺酸/酥胺酸(serine/threonine)蛋白質激酶。在目前的研究當中，我們發現BGLF4 蛋白激酶不但可以降低病毒BMRF1 活化子轉活化能力，也可以透過活化病毒BZRF1(Zta) 對溶裂期複製起點域中BHLF1 啟動子的轉活化能力影響病毒基因體的複製。由於BGLF4 蛋白激酶可以和NF-kappa B 活化促進體 (enhanceosome)中蛋白UXT (Ubiquitously expressedtranscript)有交互作用，透過基因表現分析(reporter assay)也發現BGLF4 可特異性地阻斷NF-kappa B 轉活化能力，暗示著BGLF4 蛋白激酶極可能透過抑制NF-kappa B 活性促進複製起點域病毒基因體複製。因此基於目前對於轉錄和基因體複製機制交互作用的了解，我們試圖進一步探討BGLF4 蛋白激酶調節溶裂期複製起點域轉錄活性和對病毒基因體複製起始的影響。本計畫的目標為：(i) 探討BGLF4 蛋白激酶活化BZRF1 轉活化能力的機制。 (ii) 探討BGLF4 蛋白激酶抑制NF-kappa B 訊息傳遞的機制和其對病毒基因體複製的影響。 (iii) 利用EB 病毒微陣列分析研究BGLF4 蛋白激酶對於病毒轉錄系統的影響。 (iv) 測試BGLF4 蛋白激酶對病毒基因體複製相關的蛋白複合體磷酸化所造成的影響。 (v) 研究BGLF4 蛋白激酶和核孔蛋白的作用。本研究最終的目標在釐清一個具有CDK 活性的蛋白激酶如何參與並調控病毒轉錄及基因體複製，研究成果不僅可以讓我們清楚該如何控制病毒從潛伏期活化，也有助於未來更了解真核細胞調控基因體複製的機制。<br> Abstract: Epstein-Barr virus (EBV) is an oncogenic human gamma herpesvirus associated withhuman malignancies of different origins. The most unique features of EBV replication are itsability to be repeatedly reactivated from latency into lytic replication and its cascade geneexpression control. The viral lytic replication origin (oriLyt) is composed of multiple viral andcellular transactivator binding sites. Thus the transcription activity of BHLF1 promoter at thisregion is essential for the initiation of viral DNA replication. Various cellular signalingpathways appear to participate in regulating the switching of viral status from latency to lyticreplication. For example, NF-kappa B activity was found to inhibit the reactivation of severalgamma herpesviruses, through blocking the activation of viral immediate early (IE)transactivators. In addition to the interplay between viral proteins and cellular signalingpathways, the viral BGLF4 kinase appears to play an important role in regulating multipleviral and cellular factors to optimize viral replication. BGLF4 is a proline-dependentserine/threonine kinase which has cyclin-dependent kinase (CDK)-like activities. In ourcurrently ongoing project, we demonstrated that BGLF4 can down-regulate thetransactivation function of the BMRF1 and up-regulate the transactivation of BZRF1 on theoriLyt BHLF1 promoter, which is important for the initiation of oriLyt dependent DNAreplication. On the other hand, we also observed that BGLF4 interacts with the NF-kappa Benhanceosome protein UXT (ubiquitously expressed transcript). In reporter assay, BGLF4specifically blocks NF-kappa B transactivation activity, suggesting BGLF4 may promoteoriLyt DNA replication through inhibition of NF-kappa B activity. Therefore we intend tostudy the regulatory function of BGLF4 kinase on transcription activities at oriLyt and theinitiation of viral DNA replication. Additionally, because nuclear transport is involved intranscription and DNA replication regulation control, the possible interaction of BGLF4 withnuclear pore complex will also be addressed. Specific Aims of this project are (i) To identifythe mechanism involved in BGLF4-mediated upregulation of BZLF1 (Zta) transactivation, (ii)To reveal the mechanism of BGLF4 to down regulate NF-kappa B signaling and its effects onoriLyt DNA replication, (iii) To study the regulatory effects of BGLF4 on viral transcriptionprogram using viral microarray, (iv) To examine the effects of BGLF4 mediatedphosphorylation on the protein complex involved in oriLyt DNA replication, (v) To study theinteraction between BGLF4 and nuclear pore complex. The ultimate goal of this study is toreveal how a viral CDK-like enzyme contributes to viral transcription and DNA replicationregulation. The results may not only suggest future direction to control the viral reactivationfrom latency but also shed light on eukaryotic DNA replication control.