2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642688摘要：EB 病毒是普遍感染全球人口的gamma 型皰疹病毒。初次感染EB 病毒的症狀可能很輕微，然而由血清學及血清中病毒含量分析發現EB 病毒與許多人類惡性疾病有高度相關性。EB 病毒可能協同其他致癌因子促進腫瘤生長。EB 病毒感染後，環狀構造的EB 病毒基因體可以利用宿主細胞內中DNA 複製機組來進行複製，潛伏在宿主細胞內。在不同的化學物質或表面免疫球蛋白IgG 交聯刺激後，EB 病毒之極早期蛋白Zta 及Rta 的表現會活化一連串溶裂期基因的表現。在潛伏與溶裂期的轉換過程中，不同病毒蛋白間會協同作用以利於病毒基因表現及病毒DNA 的複製，並與EBV 相關疾病之致病機轉有關聯性。我們實驗室先前的研究發現EB 病毒蛋白激酶BGLF4 在調控Zta、Rta 及早期蛋白BMRF1 之轉活化能力中扮演重要的角色。BMRF1 除了是病毒DNA 聚合酶輔助因子，同時也可作為轉活化因子去調節BHLF1 啟動子或作為共同活化因子去調節BALF2(單股DNA結合蛋白)啟動子。在病毒複製的細胞中，BGLF4 的表現與BMRF1 的高度磷酸化具有相關性。試管內實驗發現BGLF4 將BMRF1 上許多位點進行磷酸化，暗示BGLF4能透過直接磷酸化來調節BMRF1 的功能。病毒與哺乳細胞之核酸複製區類似，包含導引酶、解螺旋酶、DNA 聚合酶、BMRF1 及許多附屬蛋白大型蛋白質複合體已被證實會聚集在病毒核酸複製區。我們的初步實驗結果顯示病毒的尿嘧啶醣苷酶BKRF3 可以在鹼基切除修復過程中負責將錯誤摻入的尿嘧啶移除。在病毒複製的細胞中BKRF3 會聚集在病毒DNA 複製區。因BKRF3 不具有特定的細胞核定位訊號，且其單獨轉染至細胞後表現於細胞質。我們因此將針對BKRF3 及其他病毒蛋白如何透過病毒蛋白質間相互作用網絡而聚集到病毒DNA 複製區以達到有效率的病毒DNA 複製進一步作探討。為了在完整病毒的其他病毒蛋白質存在情況下探討上述問題，我們實驗室建立EB 病毒穿梭載體系統(EBV bacmid)後並且構築特定基因剃除之病毒穿梭載體以探討不同蛋白質之特性。另外我們製備的NTU-EB 病毒全基因組微陣列技術則可以檢測病毒全基因組表現情形。本計畫的目標為: (1) 利用EB 病毒全基因組微陣列分析研究BMRF1 參與那些病毒基因之轉錄調控。(2) 研究磷酸化對於BMRF1 功能之調節。(3) 探討參與BMRF1 調節之轉錄調控的細胞內蛋白或病毒蛋白為何。(4) 研究病毒尿嘧啶醣苷酶BKRF3 在核酸複製區的特性。(5) 探討BKRF3 與其他蛋白質結集功能區域及酵素活性功能區域。<br> Abstract: Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects most of thepopulation worldwide. Primary infection of EBV may cause mild clinical symptoms.However, studies of virus specific antibodies and viral loads in patients’ sera indicate thatEBV is highly associated with various human malignancies, suggesting EBV may play apromoting role and cooperate with other oncogenic factors to accelerate tumorigenesis. Afterprimary infection, the circularized EBV genome can persist latently in the cells and replicatein S phase using cellular DNA replication machinery. Upon the stimulation of differentchemical or surface IgG cross-linking, the expression of immediate early transactivators Zta(BZLF1) and Rta (BRLF1) can turn on cascade lytic gene expression. Thus the switchbetween latent and lytic cycle and the coordination among different viral proteins arerequired for efficient viral gene expression and DNA replication, which may link to thepathogenesis of EBV associated diseases. In previous studies, we demonstrated that EBVBGLF4 kinase plays a regulatory role on Zta, Rta and BMRF1 transactivation activities.BMRF1 is the viral DNA polymerase processivity factor, and also functions as atransactivator on BHLF1 promoter and a co-activator on BALF2 (single stranded DNAbinding protein) promoter. GLF4 expression correlates with the hyperphosphorylation ofBMRF1 in virus replicating cells. BGLF4 phosphorylates BMRF1 at multiple sites ofBMRF1 in vitro, suggesting that BGLF4 may regulate BMRF1 function through directphosphorylation. Additionally, similar to mammalian DNA replication complex, a largeprotein complex including primase/helicase/DNA polymerase, BMRF1 and severalaccessory proteins, was demonstrated to associate with viral DNA replication compartment.In our preliminary data, we found viral BKRF3 uracil DNA glycosylase, which isresponsible for the removal of misincorporated uracil in base excision repair (BER), wasrecruited to the viral DNA replication compartment and associated with viral DNA. BecauseBKRF3 does not contain a putative nuclear localization signal and expresses in thecytoplasm of transfected cells, we aim to identify to the protein interacting network thatrecruits BKRF3 and other viral proteins into the replication compartment for efficient viralDNA replication. In order to address these questions in a whole virus setting, we establishedthe EBV bacmid system in our laboratory to generate specific gene knout or mutant viruses.In addition, our homemade NTU-EBV DNA microarray also provides the possibility toexamine the genomewide gene expression profiles. Specific aims of this study are (1) Toexplore BMRF1 mediated transcriptional regulation through a genomewidemicroarray screening. (2) To study the phosphorylation mediated regulation onBMRF1 function. (3) To identify cellular or viral proteins involved inBMRF1-mediated transcriptional regulation. (4) To characterize the viral uracil DNAglycosylase BKRF3 in replication complex. (5) To characterize the functional domainsresponsible for recruitment and enzyme activity of BKRF3.