Fuentes-Pananá E M, Peng R, Brewer G, Tan J, Ling P D
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Virol. 2000 Sep;74(17):8166-75. doi: 10.1128/jvi.74.17.8166-8175.2000.
EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of a cis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.
EBNA2是一种由爱泼斯坦-巴尔病毒（EBV）编码的蛋白质，它可调节EBV驱动的B细胞永生化所需的病毒和细胞基因的表达。阐明EBNA2调节病毒和细胞基因表达的机制对于理解EBV诱导的人类B细胞永生化和病毒潜伏至关重要。EBNA2通过与细胞DNA结合蛋白CBF1（RBPJk）相互作用靶向潜伏性C启动子（Cp）。Cp中的EBNA2增强子还结合另一种细胞因子，即C启动子结合因子2（CBF2），其蛋白质产物尚未确定。在Cp中的EBNA2增强子内，我们之前已经鉴定出CBF2结合所需的DNA序列，并确定该元件是EBNA2有效激活Cp所必需的。在本研究中，对CBF2活性进行了生化纯化和微量测序。测序得到的肽段与hnRNP蛋白AUF1相同。在凝胶迁移率变动分析中，针对AUF1的抗体而非针对相关hnRNP蛋白的抗体与CBF2发生反应。此外，刺激细胞环磷酸腺苷（cAMP）/蛋白激酶A（PKA）信号转导途径会导致从受刺激细胞中提取的可检测到的CBF2/AUF1结合活性增加。此外，当用激活PKA的化合物处理转染细胞或共转染表达PKA组成型活性催化亚基的质粒时，CBF2结合位点能够赋予异源启动子对EBNA2的反应性。在类似的共转染分析中，EBNA2介导的潜伏性Cp的刺激也会增加。这些结果进一步支持了CBF2在介导EBNA2反式激活中的重要作用；它们确定hnRNP蛋白AUF1是CBF2的主要成分，也是第一个证明除CBF1结合元件外能够赋予对EBNA2反应性的顺式作用序列的证据。
