Sauder C, Haiss P, Grässer F A, Zimber-Strobl U, Mueller-Lantzsch N
Institut für medizinische Mikrobiologie und Hygiene, Universitätskliniken des Saarlandes, Homburg/Saar, Germany.
J Gen Virol. 1994 Nov;75 ( Pt 11):3067-79. doi: 10.1099/0022-1317-75-11-3067.
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Trans-activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates trans-activation. To determine whether EBNA-2 also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein-protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.
爱泼斯坦-巴尔病毒（EBV）核抗原2（EBNA-2）蛋白对于EBV使人类原代B细胞永生化至关重要。EBNA-2反式激活细胞和病毒基因，如CD23、c-fgr、潜伏膜蛋白1（LMP1）和末端蛋白1（TP1）。TP1启动子和BamHI C启动子的反式激活已被详细研究，似乎是通过蛋白质-蛋白质相互作用介导的，而非EBV 1型的EBNA-2 A型直接与DNA结合。EBNA-2能够在多种细胞系中反式激活LMP基因的表达。各种报告已描绘出EBNA-2介导反式激活的LMP启动子的顺式作用元件。为了确定EBNA-2是否也通过蛋白质-蛋白质相互作用反式激活LMP启动子，我们用不同大小的LMP启动子片段进行了一系列凝胶阻滞试验和竞争实验。我们确定LMP启动子上的蛋白质结合区域在一个42 bp的片段内，该片段包含相对于LMP转录起始位点的核苷酸-135至-176。所研究的DNA片段均未表明EBNA-2通过蛋白质-蛋白质相互作用与DNA相互作用。在能清楚显示EBNA-2 A与TP1启动子结合的条件下进行凝胶阻滞试验时，未在EBNA-2阳性和EBNA-2阴性核提取物之间观察到显著差异。然而，凝胶阻滞试验中蔗糖梯度级分的分析提供了证据，表明LMP启动子结合蛋白在EBNA-2阳性细胞提取物中形成了更高分子量（M(r)）的复合物。这些复合物被去污剂破坏。我们从这些结果推断，EBNA-2阳性细胞可能确实含有与LMP启动子结合的特异性复合物，然而，这些复合物过于不稳定，无法在标准凝胶阻滞试验中检测到。
