MECHANISMS FOR EBV EBNA-2 IMMORTALIZING FUNCTION

这项研究计划的长期目标是了解 EBNA-2在潜伏性EBV的建立和维持中的作用（Epstein-Barr 病毒）感染和EBV驱动的B细胞永生化。 永生化 B淋巴细胞通过EB病毒需要病毒转录激活蛋白 FBNA-2，因此是一个有吸引力的抗病毒药物的目标。 EBNA-2 含有有效的激活剂结构域，并通过 通过与细胞DNA结合蛋白的相互作用分离结构域 CBF 1/RBPjk（C启动子结合因子1/重组信号结合 蛋白质）。 迄今为止表征的功能结构域位于 EBNA-2蛋白的羧基末端的一半，并保守在 EBNA-2型（来自EBV-1和EBV-2），包括来自EBV-1和EBV-2的EBNA-2同源物。 乳头疱疹病毒（HVP）。 有几个问题仍然没有解决。 第一、 遗传学研究表明，EBNA-2的氨基末端的一半是 也是永生化和反式激活所必需的。 九个中的四个 EBNA-2型之间的保守区位于氨基末端， 一半，并有可能提供洞察力，以确定重要的 功能域。 这项拨款申请提出了实验来测试 这一假设，并定义额外的功能域的氨基- EBNA-2的末端。 第二，EBV分离株分为两种类型，1和 2、永生化效率不同。 生物表型可以 与EBNA-2蛋白质相关。 差异的机制 1型或2型EBNA-2之间的永生化效率是未知的。 第三，最近的研究结果也表明，参与细胞 结合EBNA-2应答启动子的因子，除了CBF 1/RBPjk， 对EBNA-2的反式激活很重要。 其中一种蛋白质叫做 CBF 2（C启动子结合因子2）是这种效应的可能候选者。 该提案中的实验旨在表征和克隆这种 因子 本提案的具体目标是审查 EBNA-2的作用和是：1）确定氨基- 通过保守区1-4的突变分析， 测试突变体的反式激活和永生化功能， 试图确定保守区1-4与 2）确定减少细胞蛋白的fa机制， 通过构建2型EBV观察到的永生化效率 1型和2型EBNA-2嵌合体，和3）鉴定和表征 EBNA-2增强子结合蛋白CBF 2通过测量CBF 2对EBNA-2增强子结合蛋白CBF 2表达的影响来测定。 EBNA-2反式激活，检查CBF 1之间间距的影响 和EBNA-2反式激活上的CBF 2结合位点，定义CBF 2结合 通过诱变定位，并纯化和克隆CBF 2。 完成 这些具体目标将提供有关EBNA-2功能的新信息 及其在EBV转化中的作用，并可能提供深入了解 细胞转化的一般原理。 了解这些 生物化学途径将允许抗EBV的合理设计， 抗癌剂。

The long-term goal of this research program is to understand the role of EBNA-2 in the establishment and maintenance of latent EBV (Epstein-Barr virus) infection and EBV-driven B cell immortalization. Immortalization of B lymphocytes by EBV requires the viral transcriptional activator protein FBNA-2 and is therefore an attractive target for anti-viral agents. EBNA-2 contains a potent activator domain and targets to promoters through a separate domain by an interaction with the cellular DNA binding protein CBF1/RBPjk (C promoter binding factor 1/recombination signal binding protein). The functional domains characterized so far reside in the carboxy-terminal half of the EBNA-2 protein and are conserved among the EBNA-2 types (from EBV-1 and EBV-2), including the EBNA-2 homologue from herpesvirus papio (HVP). Several questions still remain unsolved. First, genetic studies have indicated that the amino-terminal half of EBNA-2 is also required for immortalization and transactivation. Four of nine conserved regions between the EBNA-2 types reside in the amino-terminal half and are likely to provide insight towards identification of important functions domains. This grant application proposes experiments to test this hypothesis and define additional functional domains in the amino- terminal half of EBNA-2. Second, EBV isolates fall into two types, 1 and 2, that differ in immortalizing efficiency. The biological phenotype can be mapped to the EBNA-2 protein. The mechanism for differences in immortalizing efficiency between type 1 or type 2 EBNA-2 is not known. Third, recent findings have also indicated that participation of cellular factors that bind to EBNA-2 responsive promoters, other than CBF1/RBPjk, are important for EBNA-2 transactivation. One of these proteins called CBF2 (C promoter binding factor 2) is a likely candidate for this effect. Experiments in this proposal are aimed at characterizing and cloning this factor. The specific aims of this proposal focus on examining the mechanism of action of EBNA-2 and are: 1) To identify functional domains in the amino- terminal half of EBNA-2 by mutational analysis of conserved regions 1-4 and testing the mutants for transactivation and immortalization function and seeks to identify direct interactions of conserved regions 1-4 with cellular proteins; 2) To identify fa mechanism for the reduced immortalizing efficiency observed with the type 2 EBV by constructing type 1 and type 2 EBNA-2 chimeras, and 3) To identify and characterize the EBNA-2 enhancer binding protein CBF2 by measuring the effect of CBF2 on EBNA-2 transactivation, examining the effect of spacing between the CBF1 and CBF2 binding sites on EBNA-2 transactivation, defining the CBF2 binding site through mutagenesis, and purification and cloning of CBF2. Completion of these specific aims will give new information about how EBNA-2 functions and its role in EBV transformation, and may provide insight into the general principles of cellular transformation. Knowledge of these biochemical pathways will allow for the rational design of anti-EBV and anti-cancer agents.