Zimber-Strobl U, Kremmer E, Grässer F, Marschall G, Laux G, Bornkamm G W
Institut für Klinische Molekularbiologie und Tumorgenetik, GSF, München, Germany.
EMBO J. 1993 Jan;12(1):167-75. doi: 10.1002/j.1460-2075.1993.tb05642.x.
The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter.
爱泼斯坦-巴尔病毒蛋白EBNA2作为细胞和病毒基因的转录激活因子，在EBV使人类原代B细胞永生化过程中发挥关键作用。我们之前已经表明，EBNA2可反式激活潜伏膜抗原LMP、TP1和TP2的启动子。选择TP1基因的启动子作为模型系统来研究EBNA2介导的反式激活的分子机制。为了鉴定EBNA2依赖性顺式作用元件，在有无EBNA2表达载体的情况下，将各种TP1启动子-报告基因构建体转染到已建立的B细胞系BL41-P3HR1中。我们能够确定相对于TP1 RNA起始位点在-258至-177之间的一个81 bp的EBNA2反应区域。该元件无论何种方向均可发挥作用，并能在异源启动子上介导EBNA2依赖性反式激活。电泳迁移率变动分析显示，EBNA2反应元件序列形成了三种特异性蛋白质-DNA复合物。其中两种不是细胞类型特异性的，但第三种仅在EBNA2阳性细胞提取物中检测到。在存在EBNA2特异性单克隆抗体的情况下进行凝胶迁移分析表明，EBNA2是第三种复合物的一个组分。因此，这些实验证明EBNA2与TP1启动子的一个EBNA2反应性顺式元件相互作用。
