Ruf I K, Sample J
Program in Viral Oncogenesis and Tumor Immunology, Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Virol. 1999 Oct;73(10):7943-51. doi: 10.1128/JVI.73.10.7943-7951.1999.
During the restricted programs of Epstein-Barr virus (EBV) latency in EBV-associated tumors and a subpopulation of latently infected B cells in healthy EBV carriers, transcription of the EBV nuclear antigen 1 (EBNA-1) gene is mediated by the promoter Qp. Previously, two noncanonical E2F binding sites were identified within Qp. The role of E2F in the regulation of Qp, however, has been controversial and is undefined. Here we demonstrate that an E2F factor(s) within Burkitt lymphoma (BL) cells binds to a G/C-rich element [GGCG(C/G)] within the previously identified binding sites in Qp and prototypical E2F response elements. Furthermore, Qp-driven reporter gene expression could be efficiently repressed through either E2F binding site by the tumor suppressor pRb, a potent transcriptional repressor targeted to promoters during G(0) and the early G(1) phase of the cell cycle via its interaction with E2F; a mutant pRb (pRb(706)) lacking E2F binding capability was unable to repress Qp. However, we did not observe cell cycle variation in the expression of either EBNA-1 mRNA or protein in exponentially growing BL cells, consistent with previous predictions that Qp is constitutively active in these cells and with the extremely long t(1/2) of EBNA-1. By contrast, within G(0)/G(1) in growth-arrested BL cells, EBNA-1 mRNA levels were twofold lower than in S phase, similar to the two- to eightfold differences in cell cycle expression of some cyclin mRNAs. Thus, although regulation of Qp is coupled to the cell cycle, this clearly has no impact on the level of EBNA-1 expressed in proliferating cells. We conclude, therefore, that the most important contribution of E2F to the regulation of Qp is to direct the pRb-mediated suppression of EBNA-1 expression within resting B cells, the principal reservoir of latent EBV. This would provide a means to restrict unneeded and potentially deleterious expression of EBNA-1 in a nonproliferating cell and to coordinate the activation of EBNA-1 expression necessary for EBV genome replication and maintenance upon reentry of the cell cycle in response to proliferative signals.
在与EB病毒（EBV）相关的肿瘤以及健康EBV携带者中潜伏感染B细胞亚群的EBV潜伏受限程序期间，EBV核抗原1（EBNA-1）基因的转录由启动子Qp介导。此前，在Qp内鉴定出两个非典型E2F结合位点。然而，E2F在Qp调控中的作用一直存在争议且尚未明确。在此，我们证明伯基特淋巴瘤（BL）细胞内的一种E2F因子与Qp中先前鉴定的结合位点以及典型E2F反应元件内富含G/C的元件[GGCG(C/G)]结合。此外，肿瘤抑制因子pRb可通过任一E2F结合位点有效抑制Qp驱动的报告基因表达，pRb是一种强效转录抑制因子，在细胞周期的G(0)期和早期G(1)期通过与E2F相互作用靶向启动子；缺乏E2F结合能力的突变型pRb（pRb(706)）无法抑制Qp。然而，我们未在指数生长的BL细胞中观察到EBNA-1 mRNA或蛋白表达的细胞周期变化，这与先前关于Qp在这些细胞中组成性激活以及EBNA-1极长半衰期的预测一致。相比之下，在生长停滞的BL细胞的G(0)/G(1)期内，EBNA-1 mRNA水平比S期低两倍，类似于某些细胞周期蛋白mRNA在细胞周期表达中的两到八倍差异。因此，尽管Qp的调控与细胞周期相关，但这显然对增殖细胞中EBNA-1的表达水平没有影响。所以，我们得出结论，E2F对Qp调控的最重要贡献是指导pRb介导的静止B细胞（潜伏EBV的主要储存库）中EBNA-1表达的抑制。这将提供一种手段来限制非增殖细胞中EBNA-1不必要且可能有害的表达，并在细胞周期响应增殖信号重新进入时协调EBV基因组复制和维持所需的EBNA-1表达激活。
