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EBV的建立和维持中的作用(Epstein-Barr 病毒)感染和EB病毒驱动的B细胞永生化。永垂不朽 EB病毒感染的B淋巴细胞需要病毒转录激活蛋白 FBNA-2，因此是抗病毒药物的一个有吸引力的目标。EBNA-2 包含一个有效的激活器域，并通过 通过与细胞DNA结合蛋白的相互作用来分离结构域 CBF1/RBPjk(C启动子结合因子1/重组信号结合 蛋白质)。到目前为止描述的功能域驻留在 EBNA-2蛋白的羧基末端的一半，在 EBNA-2类型(来自EBV-1和EBV-2)，包括来自 乳头疱疹病毒(HVP)。有几个问题仍然没有得到解决。第一, 遗传学研究表明，EBNA-2的氨基末端一半是 也是永生和转化所必需的。九次中四次 EBNA-2型之间的保守区位于氨基末端 一半，并可能提供对识别重要的 功能域。这项拨款申请提出了一些实验，以测试 这一假设并定义了氨基中的附加功能结构域- EBNA-2的末端半部。其次，EBV分离株分为两种类型，1和 2、长生不老效率不同。生物表型可以 被定位于EBNA-2蛋白。中美之间差异的机制 1型或2型EBNA-2之间的永生效率尚不清楚。 第三，最近的发现也表明，细胞的参与 结合EBNA-2反应启动子的因子，而不是CBF1/RBPjk， 对EBNA-2反式激活很重要。其中一种蛋白质叫做 CBF2(C启动子结合因子2)可能是这种作用的候选基因。 这项提议中的实验旨在描述和克隆这一点 因素。 这项建议的具体目标集中在研究 EBNA-2和ARE的作用：1)识别氨基-2的功能结构域 EBNA-2末端1-4保守区突变分析 测试突变体的反式激活和永生化功能 寻求确定保守区1-4与 细胞蛋白质；2)确定FA减少的机制 通过构建类型观察到2型EBV的永生化效率 1和2型EBNA-2嵌合体，以及3)鉴定和表征 EBNA-2增强子结合蛋白CBF2通过检测CBF2对细胞周期的影响 EBNA-2反式激活，检测CBF1之间的间距的影响 和EBNA-2反式激活上的CBF2结合位点，定义CBF2结合 对CBF2进行诱变、纯化和克隆。完成 将提供有关EBNA-2如何发挥作用的新信息 以及它在EBV转型中的作用，并可能提供对 细胞转化的一般原理。了解这些知识 生化途径将允许合理设计抗EBV和 抗癌药物。

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.