Johannsen E, Koh E, Mosialos G, Tong X, Kieff E, Grossman S R
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
J Virol. 1995 Jan;69(1):253-62. doi: 10.1128/JVI.69.1.253-262.1995.
Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.
爱泼斯坦 - 巴尔病毒（EBV）潜伏膜蛋白1（LMP - 1）癌基因的表达受EBV核蛋白2（EBNA - 2）反式激活因子调控。已知EBNA - 2与细胞DNA结合蛋白Jκ相互作用，并被招募至含有GTGGGAA Jκ识别序列的启动子。最小的EBNA - 2应答性LMP - 1启动子包含一个Jκ结合位点，我们现在表明该位点发生突变，导致Jκ无法结合，会使EBNA - 2应答性降低60%。为了鉴定与LMP - 1 EBNA - 2应答元件（E2RE）相互作用的其他因子，一个 - 236 / - 145最小E2RE在电泳迁移率变动分析中用作探针。先前已鉴定的因子Jκ、PU.1和AML1与LMP - 1 E2RE结合，还有其他六个未鉴定的因子（LBF2至LBF7）。为每个因子绘制了结合位点图谱。LBF4具有B细胞和T细胞特异性，通过甲基化干扰显示它识别PU.1的GGAA核心序列。LBF4的分子量为105 kDa，可能与PU.1无关。仅在上皮细胞系中发现LBF2，而LBF3、LBF5、LBF6和LBF7不具有细胞类型特异性。AML1或LBF4结合位点的突变对EBNA - 2反式激活没有影响，而PU.1结合位点的突变则完全消除了EBNA - 2应答。一种gst - EBNA - 2融合蛋白特异性地从核提取物中耗尽PU.1，并在体外结合翻译的PU.1，为EBNA - 2与PU.1的直接相互作用提供了生化证据。因此，EBNA - 2对LMP - 1启动子的反式激活依赖于与至少两种不同的序列特异性DNA结合蛋白Jκ和PU.1的相互作用。LBF3、LBF5、LBF6或LBF7可能也参与其中，因为它们的结合位点也对EBNA - 2应答有贡献。
