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

项目简介：eb病毒（EBV）能在体外高效永生化人B细胞，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细胞永生的细胞过程中的作用。目的1。探讨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EBNA2全长有显著的负向影响。EBNA2保守区（CR） 1和2的特异性突变，对应残基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，它们也不能寡聚。我们将在基因互补试验中测试这些突变体维持B细胞永生的能力。功能分析将在ebv阳性的B细胞中进行，以确定这些突变EBNA2诱导LMP1的能力。qRT-PCR将用于检测CR1和/或CR2是否介导全局或基因特异性EBNA2活性。目标2。确定ebna - lp介导的PML NBs中Sp100的移位机制，以及这如何促进EBNA2的共激活。Sp100氨基酸残基3-152介导二聚化、PML NB定位以及与ena - lp的相互作用。为了确定ena - lp如何重新定位PML NBs中的Sp100，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检测，以确定介导自结合、ena - 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启动子上的特异性修饰（如甲基化）将通过ChIP测定。相关性：靶向寡聚化的EBNA2功能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ena - 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。

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.