Sjöblom A, Yang W, Palmqvist L, Jansson A, Rymo L
Department of Clinical Chemistry and Transfusion Medicine, Göteborg University, Sahlgrenska University Hospital, Gothenburg, Sweden.
J Virol. 1998 Feb;72(2):1365-76. doi: 10.1128/JVI.72.2.1365-1376.1998.
The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a viral oncogene whose expression is regulated by both viral and cellular factors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LMP1 expression in human B cells, and several EBNA2 response elements have been identified in the promoter regulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMP response element (ATF/CRE) site in LRS is involved in EBNA2 responsiveness. We now establish the importance of the ATF/CRE element by mutational analysis and show that both EBNA2-dependent activation and EBNA2-independent activation of the promoter occur via this site but are mediated by separate sets of factors. An electrophoretic mobility shift assay (EMSA) with specific antibodies showed that the ATF-1, CREB-1, ATF-2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun heterodimers whereas the Sp1 and Sp3 factors bind to an adjacent Sp site. Overexpression of ATF-1 and CREB-1 in the cells by expression vectors demonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 promoter in an EBNA2-independent manner. The homodimers of ATF-2 and c-Jun did not significantly stimulate promoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absence of EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protein. Evidence for a direct interaction between the ATF-2/c-Jun heterodimeric complex and EBNA2 was obtained by EMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactivation via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer. EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimeric complex between the ATF-1 and CREB-1 factors.
爱泼斯坦-巴尔病毒（EBV）潜伏膜蛋白1（LMP1）是一种病毒癌基因，其表达受病毒和细胞因子调控。EBV核抗原2（EBNA2）是人类B细胞中LMP1表达的有效反式激活因子，并且在启动子调控序列（LRS）中已鉴定出多个EBNA2反应元件。我们之前已表明，LRS中的一个激活转录因子/环磷酸腺苷反应元件（ATF/CRE）位点参与EBNA2反应性。我们现在通过突变分析确定了ATF/CRE元件的重要性，并表明启动子的EBNA2依赖性激活和EBNA2非依赖性激活均通过该位点发生，但由不同的因子介导。用特异性抗体进行的电泳迁移率变动分析（EMSA）表明，ATF-1、CREB-1、ATF-2和c-Jun因子以ATF-1/CREB-1和ATF-2/c-Jun异二聚体的形式结合到该位点，而Sp1和Sp3因子则结合到相邻的Sp位点。通过表达载体在细胞中过表达ATF-1和CREB-1表明，这些因子的同二聚体以及异二聚体形式以EBNA2非依赖性方式反式激活LMP1启动子。ATF-2和c-Jun的同二聚体并未显著刺激启动子活性。相反，ATF-2/c-Jun异二聚体在不存在EBNA2时仅有轻微的刺激作用，但与该蛋白共表达时可诱导LMP1启动子的强烈反式激活。通过EMSA和共免疫沉淀实验获得了ATF-2/c-Jun异二聚体复合物与EBNA2之间直接相互作用的证据。因此，我们的结果表明，EBNA2通过ATF/CRE位点诱导的反式激活是通过EBNA2与ATF-2/c-Jun异二聚体之间的直接接触发生的。另一方面，通过该位点的EBNA2非依赖性启动子激活由ATF-1和CREB-1因子之间的异二聚体复合物介导。
