Characterization of the CBF2 binding site within the Epstein-Barr virus latency C promoter and its role in modulating EBNA2-mediated transactivation.
Characterization of the CBF2 binding site within the Epstein-Barr virus latency C promoter and its role in modulating EBNA2-mediated transactivation.
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Epstein-Barr 病毒潜伏期 C 启动子内 CBF2 结合位点的表征及其在调节 EBNA2 介导的反式激活中的作用。
DOI：
10.1128/jvi.72.1.693-700.1998
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发表时间：
1998
影响因子：
5.4
通讯作者：
Ling,PD
中科院分区：
文献类型：
--
作者：
Fuentes-Pananá,EM;Ling,PD
EB病毒（EBV）EBNA 2蛋白是调节病毒和细胞基因表达的转录激活因子，并且对于EBV驱动的B淋巴细胞永生化也是必需的。病毒潜伏期C启动子（Cp）中的EBNA 2应答增强子结合两种细胞因子CBF 1和CBF 2。CBF 2蛋白对Cp增强子功能的确切作用目前尚不清楚。CBF 2似乎不与EBNA 2相互作用，仅在Cp EBNA 2增强子的-339和-368位置之间的CBF 1下游结合。在该区域内，可以发现8-bp序列CAGTGCGT，并且类似的序列也位于其他EBNA 2响应性启动子中CBF 1结合位点的下游。先前的研究表明，该序列中的突变和甲基化影响EBNA 2的反应性。为了研究CBF 2结合的要求，我们合成了一系列携带跨越保守核心序列和外侧侧翼序列的双颠换突变的寡核苷酸。令人惊讶的是，在5′端紧邻的4个碱基中的保守核心序列之外的突变，GGTT，对CBF 2结合具有最有害的影响。保守核心的突变具有梯度效应，5′端附近的突变对CBF 2结合的影响最大。此外，还测量了CBF 2与LMP-1、LMP-2和CD 23启动子结合的亲和力。这些启动子含有保守的核心，但缺少5′侧翼的GGTT基序，并且与CBF 2弱结合或根本不结合。使用含有CBF 2突变体结合位点的Cp报告质粒，我们还能够表明，在较低剂量的EBNA 2下，Cp反式激活需要功能性CBF 2结合位点，但较高剂量的EBNA 2反式激活CBF 2突变体启动子至野生型水平的40%。这些试验表明CBF 2对EBNA 2介导的病毒潜伏期Cp的反式激活很重要。此外，发现CBF 2活性与27和33 kDa的两种多肽相关。
The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that regulates viral and cellular gene expression and is also essential for EBV-driven immortalization of B lymphocytes. The EBNA2-responsive enhancer in the viral latency C promoter (Cp) binds two cellular factors, CBF1 and CBF2. The precise role of the CBF2 protein for Cp enhancer function is presently unclear. CBF2 does not appear to interact with EBNA2 and binds just downstream of CBF1 between positions −339 and −368 in the Cp EBNA2 enhancer. Within this region an 8-bp sequence, CAGTGCGT, can be found, and a similar sequence is also located downstream of CBF1 binding sites in other EBNA2-responsive promoters. Previous studies have indicated that mutations and methylation in this sequence affect EBNA2 responsiveness. To investigate the requirements for CBF2 binding, we synthesized a series of oligonucleotides carrying double transversion mutations spanning both the conserved core sequence and outside flanking sequences. Surprisingly, mutations outside of the conserved core sequence in 4 bases immediately flanking the 5′ end, GGTT, had the most deleterious effect on CBF2 binding. Mutations in the conserved core had a gradient effect, with those near the 5′ end having the most deleterious effects on CBF2 binding. In addition, the affinities of CBF2 for binding to the LMP-1, LMP-2, and CD23 promoters were also measured. These promoters contain the conserved core but lack the 5′ flanking GGTT motif and bound CBF2 weakly or not at all. Using Cp reporter plasmids containing CBF2 mutant binding sites, we were also able to show that at lower doses of EBNA2, Cp transactivation required a functional CBF2 binding site but that higher doses of EBNA2 transactivated CBF2 mutant promoters to 40% of wild-type levels. These assays indicate that CBF2 is important for EBNA2-mediated transactivation of the viral latency Cp. In addition, CBF2 activity was found to be associated with two polypeptides of 27 and 33 kDa.