Contributions of Epstein-Barr Virus EBNA2 and EBNA-LP to B cell immortalization

项目摘要：Epstein-Barr病毒(EBV)在体外有效地使人类B细胞永生，这需要 病毒蛋白EBNA2和EBNA-LP的表达。EBNA2是病毒和细胞基因的反式激活因子 表情。EBNA-LP是EBNA2的基因特异性共激活因子，它上调主要 病毒癌蛋白LMP1。我们实验室的广泛目标是了解EBNA2和EBNA-LP在 调节促进B细胞永生化的细胞过程。目的1.研究EBNA2的作用 氨基酸残基1-58参与转录激活和B细胞永生化。EBNA2氨基酸残基 1-58对全长EBNA2有显性负效应。EBNA2中的特定突变是保守的 对应于残基1-58的区域(CR)1和2导致缺陷的EBNA2同源齐聚。我们 获得了功能缺陷的突变体，这些突变体也不能齐聚。我们将测试这些 基因互补分析中的突变体保持B细胞永生化的能力。功能性 将在EBV阳性的B细胞中进行检测，以确定这些突变的EBNA2诱导 LMP1。QRT-PCR将用于测试CR1和/或CR2是否介导全局或基因特异性EBNA2 活动。目的2.确定EBNA-LP介导的SplOO从PML NBS置换的机制， 以及这如何有助于EBNA2的共同激活。Spl00氨基酸残基3-152参与二聚反应， PML Nb定位，以及与EBNA-LP的相互作用。确定EBNA-LP如何重新本地化SplOO 在PML NBS中，SplOO在残基1-2之间引入了连续15个氨基酸缺失。 150.突变体将用于Co-IP和IF检测，以确定介导自 结合，EBNA-LP结合和PML NB定位。EBNA-LP、SplOO和EBNA2的关联， 以及LMP1启动子上的特定修饰(例如甲基化)将由CHIP确定 化验。相关性：靶向齐聚作用的EBNA2小分子抑制剂可能是一个富有成效的药物 EB病毒相关癌症的治疗方法。我们将在中阐明EBNA-LP的机制贡献 B细胞永生化以及SplOO的正常作用，特别是与自身免疫性疾病有关的作用。

Project Summary: Epstein-Barr Virus (EBV) efficiently immortalizes human B cells in vitro and this requires expression of the viral proteins EBNA2 and EBNA-LP. EBNA2 is a transactivator of viral and cellular gene expression. EBNA-LP is a gene-specific coactivator of EBNA2, which up-regulates expression of the major viral oncoprotein LMP1. The broad objective of our lab is to understand the role of EBNA2 and EBNA-LP in modulating cellular processes that promote B cell immortalization. Aim 1. Investigate the role of EBNA2 amino acid residues 1-58 in transcription activation and B cell immortalization. EBNA2 amino acid residues 1-58 have a dominant negative effect on full length EBNA2. Specific mutations within EBNA2 conserved regions (CR) 1 and 2, corresponding to residues 1-58, result in defective EBNA2 homo-oligomerization. We have obtained functionally-deficient mutants which are also unable to oligomerize. We will test these mutants in genetic complementation assays for their ability to maintain B cell immortalization. Functional assays will be performed in EBV-positive B cells to determine the ability of these mutant EBNA2 to induce LMP1. qRT-PCR will be used to test whether CR1 and/or CR2 mediate global or gene-specific EBNA2 activity. Aim 2. Determine the mechanism of EBNA-LP-mediated displacement of SplOO from PML NBs, and how this contributes to EBNA2 coactivation. SplOO amino acid residues 3-152 mediate dimerization, PML NB localization, and interactions with EBNA-LP. To determine how EBNA-LP re-localizes SplOO out of PML NBs, consecutive 15 amino acid deletions have been introduced into SplOO between residues 1- 150. The mutants will be used in Co-IP and IF assays to define critical SplOO residues that mediate self- association, EBNA-LP binding and PML NB localization. The association of EBNA-LP, SplOO, and EBNA2, as well as specific modifications (e.g. methylation) on the LMP1 promoter will be determined by ChIP assays. Relevance: Small molecule inhibitors of EBNA2 function that target oligomerization may be a fruitful therapeutic approach for EBV-related cancers. We will clarify the mechanistic contributions of EBNA-LP in B cell immortalization, as well as the normal role of SplOO, especially as related to autoimmune diseases.