REGULATION OF REPLICATION AND LATENCY BY EBV EBNAS

EB病毒（EBV），一种人类疱疹病毒，建立了一个潜伏的 B淋巴细胞感染。 这些细胞被病毒永生化， 能够在培养物中持续增殖。 EB病毒 传统上与两种恶性疾病有关， 淋巴瘤和鼻咽癌。 最近，对EBV的认识 数据显示， 更清楚地将EBV与霍奇金淋巴瘤的一个子集联系起来， 器官移植受者EB病毒相关淋巴瘤的发生 艾滋病患者。 潜伏感染的B中只有有限数量的EBV基因表达 细胞 本提案中描述的研究重点是两个方面的作用， 其中，EBNA-l和EBNA-2，在建立和维持 潜伏期和永生化B细胞生长的建立 表型。EBNA-I在功能上是多效性的，有助于细胞的增殖。 调节其自身的表达，并且是唯一需要的病毒蛋白质 对于经由潜伏起点的复制，ori-P. EBNA-ls功能是 通过直接DNA结合介导。 然而， EBNA-1激活转录和ori-P复制仍然没有解决。 提出了（1）进一步表征DNA识别， EBNA-I结合所需的二聚化结构域， 分析. (2)为了确定EBNA-1反式激活的机制， 鉴定转录激活结构域。 (3)审查 EBNA-1诱导的结构变化和对ori-P复制的贡献 确定EBNA-1在复制和永生化中的作用 与细胞蛋白质的相互作用。 EBNA-ls贡献的评估（如果 任何）与R细胞永生化是相关的，因为EBNA-1/ori-P 组合被吹捧为人类的非整合载体系统， 基因治疗 EBNA-2是EBV诱导的永生化所必需的。 EBNA-2上调 EBNA和LMP家族潜伏基因的转录， 改变特定B细胞基因的表达。建议（4） 表征转录激活所需的EBNA-2结构域， 包括与细胞相互作用的结构域的永生化 靶向蛋白，CBF 1;与细胞相互作用的结构域 转录复合物，以及目前未知功能的结构域， 含有进化上保守的氨基酸基序。 在最后的具体 目的（5）靶向EBNA-2至应答启动子的细胞CBF 1蛋白 CBF 1在未感染细胞中的作用 考察

Epstein-Barr virus (EBV), a human herpesvirus, establishes a latent infection in B lymphocytes. These cells are immortalized by the virus and become capable of continuous proliferation in culture. EBV has traditionally been associated with two malignant diseases, Burkitt's lymphoma and nasopharyngeal carcinoma. Recently, awareness of EBVs potential to cause lymphoproliferative disease has been heightened by data more clearly associating EBV with a subset of Hodgkin's lymphomas and by the development of EBV associated lymphomas in organ transplant recipients and AIDS patients. Only a limited number of EBV genes are expressed in latently infected B cells. The research described in this proposal focuses on the role of two of them, EBNA-l and EBNA-2, in the establishment and maintenance of latency and in the establishment of the immortalized B cell growth phenotype. EBNA-l is functionally pleiotropic, contributing to the regulation of its own expression and being the sole viral protein required for replication via the latency origin, ori-P. EBNA-ls function is mediated through direct DNA binding. However the mechanisms by which EBNA-l activates transcription and ori-P replication remain unresolved. It is proposed (l) To further characterize the DNA recognition and dimerization domains that are required for EBNA-l binding by mutational analysis. (2) To determine the mechanism of EBNA-l transactivation by identifying a transcriptional activation domain(s). (3) To examine the contribution to ori-P replication of EBNA-l induced structural changes and determine the role in replication and immortalization of EBNA-l interaction with cell proteins. Assessment of EBNA-ls contribution (if any) to R cell immortalization is pertinent since the EBNA-l/ori-P combination is being touted as a non-integrating vector system for human gene therapy. EBNA-2 is essential for EBV induced immortalization. EBNA-2 up-regulates transcription of both the EBNA and LMP families of latency genes and also alters expression of specific B cell genes. It is proposed to (4) Characterize the domains of EBNA-2 required for transactivation and immortalization including the domain for interaction with the cellular targeting protein, CBF1; the domain for interaction with the cell transcription complex, and the domains of presently unknown function that contain evolutionarily conserved amino acid motifs. In the final specific aim (5) the cell CBF1 protein that targets EBNA-2 to responsive promoters will be characterized and the role of CBF1 in the uninfected cell examined.