THE EPSTEIN-BARR-VIRUS IMMORTALIZING PROTEIN EBNA-2 IS TARGETED TO DNA BY A CELLULAR ENHANCER-BINDING PROTEIN
THE EPSTEIN-BARR-VIRUS IMMORTALIZING PROTEIN EBNA-2 IS TARGETED TO DNA BY A CELLULAR ENHANCER-BINDING PROTEIN
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DOI：
10.1073/pnas.90.20.9237
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发表时间：
1993-10-15
影响因子：
11.1
通讯作者：
HAYWARD, SD
中科院分区：
文献类型：
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作者：
LING, PD;RAWLINS, DR;HAYWARD, SD
Epstein-Barr 病毒核抗原 EBNA-2 对于 Epstein-Barr 病毒诱导的 B 细胞永生化至关重要。 EBNA-2 是一种转录激活剂，能够修饰特定病毒和细胞基因的表达。然而，EBNA-2反式激活的机制一直是个谜。我们使用 CA46 类淋巴母细胞的分级提取物和细菌表达的 EBNA-2 多肽来证明 EBNA-2 通过与称为 Cp 结合因子 1 (CBF1) 的细胞 DNA 结合蛋白相互作用，靶向 Epstein-Barr 病毒潜伏 C 启动子 (Cp)。含有 EBNA-2 氨基酸 252-425 的谷胱甘肽 S-转移酶-EBNA-2 融合蛋白与 CBF1 相互作用，在电泳迁移率变动测定中产生缓慢迁移的复合物。 EBNA-2 aa 323 和 324 的突变（位于高度保守的氨基酸基序内）消除了与 CBF1 的相互作用。该突变还消除了 EBNA-2 在共转染测定中激活 Cp 的能力。通过使用电泳迁移率变动分析和 DNase I 足迹法，将 CBF1 的结合位点定位于 Cp 的残基 -359 至 -388。在最小异源启动子上游引入多个拷贝的 CBF1 结合位点赋予该启动子 EBNA-2 响应性。核心序列 CNGTGGGAA 的突变消除了 CBF1 结合，并且突变序列无法介导 EBNA-2 反式激活。 CBF1 核心序列也出现在其他 EBNA-2 响应启动子中，表明 CBF1 可能介导细胞和病毒靶标的 EBNA-2 反式激活。
The Epstein-Barr virus nuclear antigen EBNA-2 is essential for Epstein-Barr virus-induced immortalization of B cells. EBNA-2 is a transcriptional activator capable of modifying the expression of specific viral and cellular genes. However, the mechanism of EBNA-2 transactivation has been an enigma. We used a fractionated extract of CA46 lymphoblastoid cells and bacterially expressed EBNA-2 polypeptides to demonstrate that EBNA-2 is targeted to the Epstein-Barr virus latency C promoter (Cp) through interaction with a cellular DNA binding protein designated Cp binding factor 1 (CBF1). A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay. Mutation of EBNA-2 aa 323 and 324, which lie within a highly conserved amino acid motif, abolished the interaction with CBF1. This same mutation also abolished the ability of EBNA-2 to activate the Cp in a cotransfection assay. The binding site for CBF1 was localized to residues -359 to -388 of the Cp by using an electrophoretic mobility shift assay and DNase I footprinting. Introduction of multiple copies of the CBF1 binding site upstream of a minimal heterologous promoter conferred EBNA-2 responsiveness on that promoter. Mutation of a core sequence CNGTGGGAA abolished CBF1 binding, and the mutated sequence was unable to mediate EBNA-2 transactivation. The CBF1 core sequence also occurs in other EBNA-2-responsive promoters suggesting that CBF1 may mediate EBNA-2 transactivation of both cellular and viral targets.