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

描述(由申请人提供)：项目摘要：EB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是病毒和细胞基因表达的反式激活因子。EBNA-LP是EBNA2的基因特异性共激活因子，可上调主要病毒癌蛋白LMP1的表达。我们实验室的主要目标是了解EBNA2和EBNA-LP在调节促进B细胞永生化的细胞过程中的作用。目的1.研究EBNA2氨基酸残基1-58在转录激活和B细胞永生化中的作用。EBNA2氨基酸残基1-58对全长EBNA2有显性负效应。EBNA2保守区(CR)1和2内的特定突变，对应于残基1-58，导致缺陷的EBNA2同源寡聚。我们获得了功能缺陷的突变体，这些突变体也不能齐聚。我们将在遗传互补分析中测试这些突变体保持B细胞永生化的能力。将在EBV阳性的B细胞中进行功能分析，以确定这些突变的EBNA2诱导LMP1的能力。定量逆转录聚合酶链式反应将被用来测试CR1和/或CR2是否调节整体或基因特异性EBNA2活性。目的2.确定EBNA-LP介导的Sp100从PML NBS置换的机制及其对EBNA2共激活的影响。Sp100氨基酸残基3-152介导二聚、PML NB定位以及与EBNA-LP的相互作用。为了确定EBNA-LP如何将Sp100重新定位于PML NBS之外，在Sp100残基1-150之间连续引入了15个氨基酸缺失。这些突变体将用于Co-IP和IF分析，以定义介导自结合、EBNA-LP结合和PML NB定位的关键Sp100残基。EBNA-LP、Sp100和EBNA2的关联以及LMP1启动子上的特定修饰(例如甲基化)将通过芯片分析来确定。相关性：靶向寡聚的EBNA2功能的小分子抑制剂可能是治疗EBV相关癌症的有效方法。我们将阐明EBNA-LP在B细胞永生化中的作用机制，以及Sp100的正常作用，特别是与自身免疫性疾病相关的作用。

DESCRIPTION (provided by applicant): 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 Sp100 from PML NBs, and how this contributes to EBNA2 coactivation. Sp100 amino acid residues 3-152 mediate dimerization, PML NB localization, and interactions with EBNA-LP. To determine how EBNA-LP re-localizes Sp100 out of PML NBs, consecutive 15 amino acid deletions have been introduced into Sp100 between residues 1- 150. The mutants will be used in Co-IP and IF assays to define critical Sp100 residues that mediate self- association, EBNA-LP binding and PML NB localization. The association of EBNA-LP, Sp100, 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 Sp100, especially as related to autoimmune diseases.