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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

基本信息

批准号：
7317804
负责人：
Chisaroka W Echendu
金额：
$ 2.63万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-01 至 2008-07-27
项目状态：
已结题

项目摘要

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.

项目摘要：Epstein-Barr 病毒 (EBV) 在体外有效地使人类 B 细胞永生化，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达。 EBNA-LP 是 EBNA2 的基因特异性共激活剂，可上调主要基因的表达病毒癌蛋白LMP1。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。目标 1. 研究 EBNA2 的作用转录激活和 B 细胞永生化中的氨基酸残基 1-58。 EBNA2氨基酸残基 1-58对全长EBNA2具有显着的负面影响。 EBNA2 内保守的特定突变区域 (CR) 1 和 2，对应于残基 1-58，导致 EBNA2 同源寡聚化缺陷。我们已经获得了功能缺陷的突变体，也无法寡聚化。我们将测试这些基因互补测定中的突变体维持 B 细胞永生化的能力。功能性将在 EBV 阳性 B 细胞中进行测定，以确定这些突变体 EBNA2 诱导末次月经1。 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。目标 2. 确定 EBNA-LP 介导的 SplOO 从 PML NB 置换的机制，以及这如何促进 EBNA2 共激活。 Spl00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。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残基1-之间的Sp100中 150.突变体将用于 Co-IP 和 IF 测定，以定义介导自体介导的关键 Sp100 残基。关联、EBNA-LP 结合和 PML NB 定位。 EBNA-LP、Spl00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 ChIP 确定化验。相关性：针对寡聚化的 EBNA2 功能小分子抑制剂可能是富有成效的 EBV 相关癌症的治疗方法。我们将阐明 EBNA-LP 在以下方面的机制贡献： B细胞永生化，以及Spl00的正常作用，特别是与自身免疫性疾病相关。