Functions of EBNA1 in replication & partitioning of EBV

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)是一种导致传染性单核细胞增多症的人类疱疹病毒。前瞻性流行病学研究表明，EBV与至少两种恶性肿瘤有因果关系--地方性Burkitt淋巴瘤和鼻咽癌。EBV的基因组在所有这些疾病中都是潜伏的，并作为一个核集合体维持着，每个细胞周期复制一次，并在有丝分裂时有效地分裂。了解EBV表观小体在每个细胞周期复制一次并被分割的机制，最终将产生治疗由潜伏的EBV感染引起的所有疾病的方法。这些研究的长期目标是了解病毒基因组一般是如何维持在能够引起人类疾病的功能状态的，但EBV基因组具体是如何维持在功能状态的。EBV基因组维持和分割所需的唯一病毒蛋白是EB病毒核抗原1(EBNA1)，它通过其羧基末端DNA结合域与病毒顺式元件ORIP结合。ORIP包含两组EBNA1-DS的结合位点，一个是作为复制起点的区域，另一个是当EBNA1与之结合时作为Episome维护和分割元件的区域FR。我们最近发现EBNA1的氨基末端结构域中有两个区域是AT-钩子，对应于细胞蛋白的AT-钩子，如原型AT-钩蛋白HMGA1a。证实了这一观察结果，我们已经证明了我们可以在功能上用HMGA1a的AT-挂钩取代EBNA1的N-端。在这项建议中，我们建议在三个方面扩展我们的研究： 1.研究EBNA1的AT-钩区在染色体拴系和Episome维持中的作用。 2.利用一个新的复制子和融合蛋白来了解EBNA1在复制中的功能。 3.用病毒遗传学方法确定EBNA1在EBV诱导幼稚B细胞永生化中的作用

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis. Prospective epidemiologic studies indicate that EBV is causally associated with at least two malignancies - endemic Burkitt's lymphoma, and nasopharyngeal carcinoma. EBV's genome is latent in all these diseases, and is maintained as a nuclear episome that replicates once per cell cycle, and is efficiently partitioned at mitosis. Understanding the mechanism by which EBV episomes are replicated once per cell-cycle and partitioned will ultimately yield therapies against all diseases caused by a latent EBV infection. The long-range goal of these studies is to understand how viral episomes in general, but EBV genomes in specific, are maintained in a functional state with the capacity to cause human disease. The only viral protein that is required for EBV genome maintenance and partitioning is the Epstein-Barr nuclear antigen 1 (EBNA1), which binds a viral cis-element termed oriP through its carboxy-terminal DNA-binding domain. OriP contains two sets of binding sites for EBNA1 - DS, a region that serves as an origin of replication, and FR, a region that serves as an episome maintenance and partitioning element when EBNA1 is bound to it. We have recently discovered that the amino-terminal domain of EBNA1 has two regions within it that are AT-hooks, corresponding to the AT-hooks of cellular proteins such as the prototypic AT-hook protein HMGA1a. Confirming this observation, we have demonstrated that we can functionally replace the N-terminus of EBNA1 with the AT-hooks of HMGA1a. In this proposal we propose to extend our studies in three areas: 1. To characterize the roles of the AT-hook regions of EBNA1 in chromosome tethering and episome maintenance. 2. To utilize a novel replicon and fusion protein to understand the function of EBNA1 in replication. 3. To use viral genetics to determine the functions of EBNA1 in the immortalization of naive B-cells by EBV