BMRF1的磷酸化對EB病毒複製之影響

Abstract

EB病毒（Epstein-Barr Virus）為人類γ型疱疹病毒的一員，此病毒已經知與許多惡性腫瘤有密切相關性，包括免疫缺失所引起的B細胞瘤、T細胞淋巴瘤、霍金氏症及鼻咽癌等疾病。EB病毒感染宿主後可進入潛伏期，當EB病毒進入溶裂期時，BMRF1是病毒DNA複製時必需的七種溶裂期蛋白質之一。BMRF1為DNA聚合酶輔助因子（DNA polymerase processivity factor），可在病毒DNA複製叉上透過與EB病毒聚合酶BALF5的結合，而增加BALF5結合到DNA上的穩定度。BMRF1也可藉由調控細胞轉錄因子Sp1/ZBP-89以轉活化（transactivate）溶裂期複製起始區域（oriLyt）之BHLF1啟動子，或是與極早期蛋白質Zta更進一步協同活化（synergistic activation）BHLF1啟動子。本實驗室先前的研究已知BMRF1會被EB病毒的Serine/Threonine蛋白質激酶（protein kinase）BGLF4所磷酸化，並抑制BMRF1對BHLF1啟動子的轉活化能力，因此本研究探討磷酸化對於BMRF1功能之影響。實驗結果發現BMRF1上10個Ser-Pro-或Thr-Pro-殘基點突變成無法被磷酸化的Ala或Val時，BMRF1(8A2V)較野生型BMRF1更能抵抗BGLF4對其轉活化能力之抑制作用，而且BMRF1(8A2V)轉活化BHLF1啟動子之能力略優於野生型BMRF1。同時也觀察到BGLF4是透過磷酸化BMRF1而降低其轉活化能力，而非影響BMRF1蛋白質表現量。為探討其中可能的作用機制，以共同免疫沉澱觀察到BGLF4可能會透過激酶活性或是蛋白質間的交互作用影響BMRF1與ZBP-89之交互作用。為進一步瞭解BMRF1的磷酸化對於EB病毒溶裂期複製之影響，利用Doxycycline提引的293TetER細胞，建立含有BMRF1基因剔除之EB病毒基因體的EREVp2089ΔBMRF1細胞株（亦稱MD2細胞株）。與野生型EREVp2089細胞株相比，當MD2細胞株進入溶裂期後，發現溶裂期極早期蛋白質Rta與Zta、早期蛋白質BGLF4表現正常，但晚期蛋白質VCA與gp350/220表現量顯著減少。此外也發現缺乏BMRF1基因之EB病毒不能完成溶裂期DNA複製。進一步在MD2細胞中轉染BMRF1或BMRF1(8A2V)表現質體，觀察BMRF1的磷酸化對EB病毒溶裂期之影響。實驗結果顯示表現BMRF1可使晚期蛋白質表現量回升，EB病毒也可完成溶裂期DNA複製，然而野生型BMRF1挽救溶裂期DNA複製之效率略優於BMRF1(8A2V)。在未來的實驗中，將會轉染不同功能性蛋白質區域缺失之BMRF1突變質體至MD2提引細胞內，並利用DNA微陣列技術（DNA microarray）研究BMRF1不同的功能性蛋白質區域對於EB病毒基因表現之影響。

Epstein-Barr virus (EBV) is a human gamma-herpesvirus which has been demonstrated to be highly associated with many human malignancies, such as B lymphoma, T lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinoma. BMRF1 is one of the essential proteins required for EBV lytic replication, which functions to serve as a processivity factor for EBV polymerase BALF5 in stabilized DNA binding at the viral replication fork. BMRF1 also transactivates viral early promoter BHLF1 through cellular transcription factor Sp1/ZBP-89 or synergistically activates BHLF1 promoter with EBV immediately early transactivator, Zta. In order to investigate the effects of phosphorylation on BMRF1, the residues of Ser-Pro or Thr-Pro which are the BGLF4 putative phosphorylation sites on BMRF1 were mutated to Ala or Val to generate the phosphorylation-defective mutant, BMRF1(8A2V). In this study, BMRF1(8A2V) was found more resistant to BGLF4-mediated inhibition on its transactivation activity than that of wild-type BMRF1. BGLF4 inhibited the transactivation activity of BMRF1 through BMRF1 phosphorylation, but not protein degradation. The ability of BMRF1(8A2V) to transactivate BHLF1 promoter was better than that of wild-type BMRF1. The result of co-immunoprecipitation showed that the interaction between ZBP-89 and wild-type BMRF1 or BMRF1(8A2V), was reduced in the presence of BGLF4 or kinase dead, revealing that BGLF4 may disrupt the interaction between BMRF1 and ZBP-89 through phosphorylation or protein-protein interaction. To further investigate the regulatory effects of BMRF1 phosphorylation on EBV lytic replication, EBV(ΔBMRF1) knockout system was established. The EBV(ΔBMRF1) bacmid was transfected into 293TetER cells, which harbor a tetracycline inducible Rta, to generate EREVp2089ΔBMRF1 cells (abbreviated as MD2 cells). In MD2 cells, the lytic proteins could be induced by doxycycline, but the expression of late proteins, VCA and gp350/220, was significantly reduced in immunoblotting assay. Moreover, the viral DNA replication was also impaired in MD2 cells. By compensation of wild-type BMRF1 or BMRF1(8A2V) into MD2 cells, the effects of BMRF1 phosphorylation on viral lytic replication were studied. The result showed that the complementation with wild-type BMRF1 rescued the expression of viral late protein gp350/220 and the lytic DNA replication in MD2 cells. Additionally, BMRF1(8A2V) could also compensate the viral lytic replication of MD2 cells, though to a less extent. With this system, the influences of BMRF1 mutation or functional domain deletion on lytic gene expression will be systematically studied by EBV DNA microarray analysis.