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

Epstein-Barr 病毒 EBNA2 和 EBNA-LP 对 B 细胞永生化的贡献

• 批准号: 7231100
• 负责人: Chisaroka W Echendu
• 金额: $ 3.14万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7231100
• 关键词: mutant; role

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.

描述（由申请人提供）：项目总结：EB病毒（EBV）在体外有效永生化人B细胞，这需要表达病毒蛋白EBNA 2和EBNA-LP。EBNA 2是病毒和细胞基因表达的反式激活因子。EBNA-LP是EBNA 2的基因特异性共激活物，其上调主要病毒癌蛋白LMP 1的表达。我们实验室的广泛目标是了解EBNA 2和EBNA-LP在调节促进B细胞永生化的细胞过程中的作用。目标1.研究EBNA 2氨基酸残基1-58在转录激活和B细胞永生化中的作用。EBNA 2氨基酸残基1-58对全长EBNA 2具有显性负效应。对应于残基1-58的EBNA 2保守区（CR）1和2内的特定突变导致缺陷性EBNA 2同源寡聚化。我们已经获得了功能缺陷的突变体，它们也不能寡聚化。我们将在遗传互补试验中测试这些突变体维持B细胞永生化的能力。将在EBV阳性B细胞中进行功能测定，以确定这些突变EBNA 2诱导LMP 1的能力。qRT-PCR将用于检测CR 1和/或CR2是否介导整体或基因特异性EBNA 2活性。目标2.确定EBNA-LP介导的Sp100从PML NB中置换的机制，以及这如何有助于EBNA 2共激活。Sp100氨基酸残基3-152介导二聚化、PML NB定位以及与EBNA-LP的相互作用。为了确定EBNA-LP如何将Sp100从PML NB中重新定位，在Sp100的残基1- 150之间引入了连续的15个氨基酸缺失。突变体将用于Co-IP和IF试验，以确定介导自缔合、EBNA-LP结合和PML NB定位的关键Sp100残基。EBNA-LP、Sp100和EBNA 2的相关性以及LMP 1启动子上的特异性修饰（例如甲基化）将通过ChIP测定来确定。相关性：靶向寡聚化的EBNA 2小分子抑制剂可能是EBV相关癌症的有效治疗方法。我们将阐明EBNA-LP在B细胞永生化中的机制贡献，以及Sp100的正常作用，特别是与自身免疫性疾病相关的作用。