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）有效地使人类B细胞体外永生，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达。 EBNA-LP是EBNA2的基因特异性共激活因子，它上调了主要的表达病毒癌蛋白LMP1。我们实验室的广泛目的是了解EBNA2和EBNA-LP在调节促进B细胞永生的细胞过程。目标1。研究EBNA2的作用转录激活和B细胞永生中1-58的氨基酸残基。 EBNA2氨基酸残基 1-58对全长EBNA2具有主要的负面影响。 EBNA2中保守的特定突变与残基1-58相对应的区域（CR）1和2导致EBNA2 HOMO-OLIGOMERIADE。我们已经获得了也无法寡聚的功能缺陷突变体。我们将测试这些遗传互补测定中的突变体具有维持B细胞永生化的能力。功能测定将在EBV阳性B细胞中进行，以确定这些突变体EBNA2诱导的能力 LMP1。 QRT-PCR将用于测试CR1和/或CR2是否介导全球或基因特异性EBNA2 活动。 AIM 2。确定eBNA-LP介导的Sploo位移的机理，PML NBS，以及这如何促进EBNA2共激活。 Sploo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 NBS中，已经将15个氨基酸缺失引入了残基之间的Sploo 1-- 150。突变体将用于co-IP，以及是否定义了介导自我的关键Sploo残基关联，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）将通过芯片确定测定。相关性：靶向寡聚的EBNA2功能的小分子抑制剂可能是富有成果的与EBV相关的癌症的治疗方法。我们将阐明EBNA-LP在 B细胞永生化以及Sploo的正常作用，尤其是与自身免疫性疾病有关的。