Bell A, Skinner J, Kirby H, Rickinson A
CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, United Kingdom.
Virology. 1998 Dec 5;252(1):149-61. doi: 10.1006/viro.1998.9440.
Epstein-Barr virus, a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this growth transforming infection depends on a viral promoter in BamHI W (Wp) whose regulation is poorly understood. Using Wp reporter constructs in in vitro transfection assays, we found that Wp was 11- to 190-fold more active in B cell than in non-B cell lines and that three regions of the promoter (termed UAS1, UAS2, and UAS3) contributed to transcriptional activation. The upstream regions UAS3 (-1168 to -440) and UAS2 (-352 to -264) both functioned in a cell lineage-independent manner and were together responsible for the bulk of Wp activity in non-B cells; mutational analysis indicated the importance of a YY1 binding site in UAS2 in that context. By contrast, UAS1 (-140 to -87) was B cell specific and was the key determinant of the promoter's increased activity in B cell lines. Mutational analysis of UAS1 sequences combined with in vitro bandshift assays revealed the presence of three binding sites for cellular factors in this region. When mutations that abolished factor binding in bandshift assays were introduced into a Wp reporter construct, the loss of any one of the three UAS1 binding sites was sufficient to reduce promoter activity by 10- to 30-fold in B cells. From sequence analysis, two of these appear to be novel transcription factor binding sites, whereas the third was identified as a cyclic AMP response element (CRE). Our data indicate that this CRE interacts with CREB and ATF1 proteins present in B cell nuclear extracts and that this interaction is important for Wp activity.
爱泼斯坦-巴尔病毒是一种人类γ疱疹病毒，拥有一组独特的潜伏基因，其在B细胞中的组成性表达会导致细胞生长转化。这种生长转化感染的起始取决于BamHI W（Wp）中的一个病毒启动子，但其调控机制尚不清楚。在体外转染实验中使用Wp报告基因构建体，我们发现Wp在B细胞中的活性比在非B细胞系中高11至190倍，并且启动子的三个区域（称为UAS1、UAS2和UAS3）有助于转录激活。上游区域UAS3（-1168至-440）和UAS2（-352至-264）均以细胞谱系非依赖性方式发挥作用，共同负责非B细胞中Wp活性的大部分；突变分析表明，在这种情况下，UAS2中YY1结合位点的重要性。相比之下，UAS1（-140至-87）是B细胞特异性的，是启动子在B细胞系中活性增加的关键决定因素。对UAS1序列的突变分析与体外凝胶迁移实验相结合，揭示了该区域存在三个细胞因子结合位点。当在凝胶迁移实验中消除因子结合的突变被引入Wp报告基因构建体时，三个UAS1结合位点中任何一个的缺失都足以使B细胞中的启动子活性降低10至30倍。从序列分析来看，其中两个似乎是新的转录因子结合位点，而第三个被鉴定为环磷酸腺苷反应元件（CRE）。我们的数据表明，这个CRE与B细胞核提取物中存在的CREB和ATF1蛋白相互作用，并且这种相互作用对Wp活性很重要。
