Meitinger C, Strobl L J, Marschall G, Bornkamm G W, Zimber-Strobl U
Institut für Klinische Molekularbiologie und Tumorgenetik im Forschungszentrum für Umwelt und Gesundheit, GSF, Munich, Germany.
J Virol. 1994 Nov;68(11):7497-506. doi: 10.1128/JVI.68.11.7497-7506.1994.
EBNA2 is one of the few genes of Epstein-Barr virus which are necessary for immortalization of human primary B lymphocytes. The EBNA2 protein acts as a transcriptional activator of several viral and cellular genes. For the TP1 promoter, we have shown previously that an EBNA2-responsive element (EBNA2RE) between -258 and -177 relative to the TP1 RNA start site is necessary and sufficient for EBNA2-mediated transactivation and that it binds EBNA2 through a cellular factor. To define the critical cis elements within this region, we cloned EBNA2RE mutants in front of the TP1 minimal promoter fused to the reporter gene for luciferase. Transactivation by EBNA2 was tested by transfection of these mutants in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR-1. The analysis revealed that two identical 11-bp motifs and the region 3' of the second 11-bp motif are essential for transactivation by EBNA2. Methylation interference experiments indicated that the same cellular factor in the absence of EBNA2 binds either one (complex I) or both (complex III) 11-bp motifs with different affinities, giving rise to two different specific protein-DNA complexes within the left-hand 54 bp of EBNA2RE. A third specific complex was shown previously to be present only in EBNA2-expressing cells and to contain EBNA2. Analysis of this EBNA2-containing complex revealed the same protection pattern as for complex III, indicating that EBNA2 interacts with DNA through binding of the cellular protein to the 11-bp motifs. Mobility shift assays with the different mutants demonstrated that one 11-bp motif is sufficient for binding the cellular factor, whereas for binding of EBNA2 as well as for efficient transactivation by EBNA2, both 11-bp motifs are required.
EBNA2是爱泼斯坦-巴尔病毒中少数几个对人类原代B淋巴细胞永生化所必需的基因之一。EBNA2蛋白作为几种病毒和细胞基因的转录激活因子。对于TP1启动子，我们之前已经表明，相对于TP1 RNA起始位点，在-258至-177之间的一个EBNA2反应元件（EBNA2RE）对于EBNA2介导的反式激活是必要且充分的，并且它通过一种细胞因子与EBNA2结合。为了确定该区域内的关键顺式元件，我们在与荧光素酶报告基因融合的TP1最小启动子前克隆了EBNA2RE突变体。通过在不存在和存在EBNA2表达载体的情况下将这些突变体转染到已建立的B细胞系BL41-P3HR-1中来测试EBNA2的反式激活作用。分析表明，两个相同的11碱基基序以及第二个11碱基基序的3'区域对于EBNA2的反式激活至关重要。甲基化干扰实验表明，在不存在EBNA2的情况下，相同的细胞因子以不同亲和力结合一个（复合体I）或两个（复合体III）11碱基基序，在EBNA2RE左手边的54 bp内产生两种不同的特异性蛋白质-DNA复合体。先前已表明第三种特异性复合体仅存在于表达EBNA2的细胞中并包含EBNA2。对这种含EBNA2的复合体的分析揭示了与复合体III相同的保护模式，表明EBNA2通过细胞蛋白与11碱基基序的结合与DNA相互作用。对不同突变体进行的迁移率变动分析表明，一个11碱基基序足以结合细胞因子，而对于EBNA2的结合以及EBNA2的有效反式激活，则需要两个11碱基基序。
