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

项目概述：EB病毒（EBV）在体外有效地永生化人类B细胞，这需要 病毒蛋白EBNA 2和EBNA-LP的表达。EBNA 2是病毒和细胞基因的反式激活因子 表情EBNA-LP是EBNA 2的基因特异性共激活因子，其上调主要的 病毒癌蛋白LMP 1。我们实验室的广泛目标是了解EBNA 2和EBNA-LP在以下方面的作用： 调节促进B细胞永生化的细胞过程。目标1.研究EBNA 2的作用 转录激活和B细胞永生化中的氨基酸残基1-58。EBNA 2氨基酸残基 1-58对全长EBNA 2具有显性负效应。EBNA 2内的特异性突变保守 对应于残基1-58的区域（CR）1和2导致缺陷性EBNA 2同源寡聚化。我们 已经获得了也不能寡聚化的功能缺陷突变体。我们将测试这些 突变体在遗传互补测定中维持B细胞永生化的能力。功能 将在EBV阳性B细胞中进行测定，以确定这些突变型EBNA 2诱导 LMP1。qRT-PCR将用于测试CR 1和/或CR 2是否介导全局或基因特异性EBNA 2 活动目标二。确定EBNA-LP介导的SplOO从PML NB置换的机制， 以及这如何促进EBNA 2共激活。SplOO氨基酸残基3-152介导二聚化， PML NB定位和与EBNA-LP的相互作用。为了确定EBNA-LP如何重新定位SplOO， 在PML NB中，连续15个氨基酸缺失已被引入到SplOO中残基1- 10之间。 150.突变体将用于Co-IP和IF测定，以确定介导自身免疫的关键SplOO残基。 结合、EBNA-LP结合和PML NB定位。EBNA-LP、SplOO和EBNA 2的关联， 以及LMP 1启动子上的特异性修饰（例如甲基化）将通过ChIP 测定。相关性：靶向寡聚化的EBNA 2功能的小分子抑制剂可能是一种富有成效的 EBV相关癌症的治疗方法。我们将阐明EBNA-LP在 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。

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.