EB病毒BGLF4蛋白質激酶對於BMRF1的磷酸化及功能調控之探討

Study on the Epstein-Barr virus BGLF4 Kinase-mediated Phosphorylation and Functional Regulation of BMRF1

楊珮雯(Pei-Wen Yang)

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(2008年)

https://doi.org/10.6342/NTU.2008.00884

摘要

EB病毒隸屬於人類γ型疱疹病毒。EB病毒的感染，和巴氏淋巴瘤，鼻咽癌與何杰金氏症的形成有高度相關性。在本論文中，主要是針對EB病毒的一個溶裂期蛋白BGLF4的研究。根據序列的分析，BGLF4被認為是人類疱疹病毒UL蛋白質激酶。它被證明是一個Ser/Thr蛋白質激酶，並且具有類似細胞週期蛋白質激酶CDKs的活性。病毒早期蛋白BMRF1(EA-D)是BGLF4第一個確定的病毒蛋白受質。但是BGLF4對BMRF1的磷酸化調控功能尚不清楚。BMRF1除了是病毒溶裂時期的DNA聚合酶輔助因子外，也可以轉活化溶裂期DNA複製啟始區域(oriLyt)上的BHLF1啟動子。BMRF1可以經由細胞轉錄因子Sp1或ZBP-89的反應區域，達成對BHLF1啟動子的轉活化，或是與特早期轉活化因子Zta的共同作用讓BHLF1啟動子達到最高度轉活化。此區域啟動子的活化被認為會增加溶裂期DNA複製啟始的效率。在本研究?堙A首先利用專一性抗體，偵測BGLF4在EB病毒進入溶裂期的細胞中發現，BGLF4與BMRF1同屬於病毒溶裂時期早期蛋白，它是以磷酸化蛋白的型式存在，並且主要分佈在細胞核中。BGLF4並且會與BMRF1及病毒特早期轉活化因子Zta共同表現在細胞核中病毒溶裂期DNA複製區域。進一步分析BGLF4對BMRF1磷酸化的殘基位置，及其轉活化功能的調控，發現BGLF4會磷酸化BMRF1上可能的”樞紐區域”(hinge region)中的Ser-337, Thr-344, Ser-349與Thr-355四個位置，達成磷酸化的效果。在功能調控的研究中，發現BGLF4會抑制BMRF1對BHLF1啟動子的轉活化能力，但會加強BMRF1與Zta對BHLF1啟動子之共同轉活化能力，以及Zta本身的轉活化能力。利用仿效磷酸化的突變型BMRF1的分析，進一步證明BGLF4可以藉由上述這四個殘基位置的磷酸化，加強BMRF1與Zta對BHLF1啟動子之共同轉活化能力。有趣的是BMRF1失去在這四個殘基位置上被磷酸化能力的BMRF1突變型2A2V也會受到BGLF4的抑制調控，顯示BGLF4抑制BMRF1單獨對BHLF1啟動子的轉活化能力，並非經由上述的四個殘基位置。在最後一部份的研究中，我們推測BGLF4可能只需辨識-S-P-或-T-P-序列即可對其產生磷酸化。構築在10個S-P-或-T-P-序列上都突變成無法被磷酸化的8A2V的突變株，將BMRF1上所有可能被磷酸化的位置變異，發現此突變株之轉活化能力即不被BGLF4抑制。在共同免疫沈澱反應中，更一步發現BGLF4會阻斷BMRF1 2A2V突變株與細胞中的轉錄因子ZBP-89結合的能力，但是並不會阻斷BMRF1 8A2V與ZBP-89結合。綜合上述的研究結果，我們認為BGLF4會藉由多重機制去調控BMRF1與Zta對溶裂期DNA複製啟始區域BHLF1啟動子的活化功能，以確保溶裂期DNA複製的啟始效率。此研究也提供了γ型人類疱疹病毒(gammaherpesvirus)的DNA聚合酶輔助因子上推測之樞紐區域可能的磷酸化調控。

關鍵字

EB病毒 ； BGLF4蛋白質激酶BMRF1聚合酶輔助因子 ； Zta特早期轉活化因子 ； BHLF1啟動子 ； 溶裂期DNA複製 ； 轉活化

並列摘要

Epstein-Barr virus (EBV) belongs to gamma-herpesvirus. Its infection is highly associated with the pathogenesis of human malignancies including Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), and Hodgkin’s disease (HD). One of the EBV lytic gene products, BGLF4, is what we focused on in the research. BGLF4 was identified as a herpesviral UL protein kinase based on sequence alignment. It is a Ser/Thr protein kinase, and can partially mimic the activities of cellular cyclin-dependent kinases (CDKs). The first identified BGLF4 substrate is the lytic early antigen BMRF1 (EA-D), but its impact on BMRF1 function is unclear. BMRF1 is the viral DNA polymerase processivity factor. In addition to processivity function, BMRF1 carries the transactivation activity to activate viral BHLF1 promoter, which is localized within the region of lytic replication origin (oriLyt). BMRF1 can transactivate BHLF1 promoter alone mediated by Sp1/ZBP-89 sites or synergistically function with immediate early transactivator Zta for maximal activation. The activation of BHLF1 promoter is suggested to be required for the initiation of viral lytic replication. In the current study, we characterized the expression of BGLF4 by specific antibody and further investigated the BGLF4-mediated phosphorylation and functional regulation of BMRF1. We found that BGLF4 is a viral lytic early protein, expresses as a phosphorprotein, and co-localizes with BMRF1 and origin binding protein Zta to the viral replication compartment. Four residues Ser-337, Thr-344, Ser-349 and Thr-355 located in putative hinge region of BMRF1 are mapped to be responsible for the BGLF4-induced hyperphosphorylation. In functional analyses, BGLF4 downregulates BMRF1-induced activation of BHLF1 promoter but enhances the synergistic transactivation of BMRF1 and Zta, and Zta alone on the BHLF1 promoter. By analyzing the phosphorylation-mimicking BMRF1, the phosphorylation of these residues is observed to enhance the synergy of BMRF1 and Zta. Interestingly, the phosphorylation-defective mutant of BMRF1 2A2V that was mutated in the four mapped residues, still can response to BGLF4-induced downregulation as that in the wild type BMRF1. In the final part, we observe the phosphorylation-defective BMRF1 mutant 8A2V which was mutated in all of the 10 proline-dependent phosphorylation sites (SP/TP) of BMRF1, is resistant to BGLF4-induced downregulation of transactivation activity. Moreover, the interaction of BMRF1 2A2V and ZBP-89 is decreased in the presence of BGLF4, but is not observed in BMRF1 8A2V mutant. Taken together, our findings suggest BGLF4 modulates BMRF1 and Zta mediated activation of oriLyt BHLF1 promoter through multiple mechanisms, which may ensure efficient initiation of lytic replication. The study gives an insight to the phosphorylated regulation locating in the possible hinge region of gammaherpes viral processivity factors.

並列關鍵字

Epstein-Barr virus (EBV) ； BGLF4 protein kinase ； BMRF1 processivity factor ； Zta immediate early transactivator ； BHLF1 promoter ； viral lytic replication ； transactivation

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