REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

细胞和病毒蛋白对 EBV Ori-LYT 的调节

• 批准号: 6642887
• 负责人: KENNEY C SHANNON
• 金额: $ 43.42万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6642887
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; DNA replication origin; Epstein Barr virus; binding proteins; confocal scanning microscopy; electron microscopy; fluorescence microscopy; genetic enhancer element; genetic promoter element; genetic transcription; host organism interaction; latent virus infection; p53 gene /protein; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor; tumor suppressor genes; virus DNA; virus genetics; virus infection mechanism; virus protein; virus replication

Epstein-Barr virus (EBV) infection has been associated with the development of several types of human malignancy. The horizontal transmission of EBV from host to host requires activation of the lytic origin of replication, oriLyt. Lytic EBV replication requires six vira1ly encoded replication proteins (BALF5, BMRF1, BALF2, BBLF4, BSLF1, and BBLF2/3). In addition, the immediate-early transactivator protein, BZLF1, is essential for oriLyt replication, even when the other viral replication proteins are expressed by a constitutively active promoter. Although the functional equivalent of the herpes simplex virus origin binding protein, UL9, has not been definitively identified for EBV, only two small regions in the EBV oriLyt (the "upstream" and "downstream" essential domains) are absolutely required for replication. Binding of BZLF1 to the upstream essential domain is necessary for oriLyt replication. We have recently discovered that the downstream essential domain is transcriptionally activated by the BMRF1 protein (the viral polymerase processivity factor), and shown that small mutations in oriLyt which abolish BMRF1-induced transactivation also abolish oriLyt replication. Thus, the BMRF1 protein appears to have two separate roles in oriLyt replication, serving both as the polymerase processivity factor, and a transcriptional activator. In this grant, we propose to study the regulation of oriLyt by the viral proteins, BMRF1 and BZLF1. Our specific aims are to l) define the mechanism by which BMRF1 transcriptionally activates oriLyt, 2) determine if the transcriptional activator function of BMRF1 (apart from its polymerase processivity function) is required for oriLyt replication, 3) examine the effects of BZLF1 and BMRF1 on oriLyt structure using electron microscopy, and 4) determine if EBV (like other herpesviruses) disrupts PML-associated nuclear bodies during lytic infection, and if so, whether this effect is required for oriLyt replication. We hypothesize that transcriptional activation of oriLyt by BMRF1 is required for lytic EBV replication, and that BZLF1 and/or BMRF1 serves as the functional origin- binding protein of oriLyt.

爱泼斯坦-巴尔病毒(EBV)感染与多种人类恶性肿瘤的发生发展有关。EBV在宿主之间的水平传播需要激活复制的裂解起点oriLyt。裂解性EBV复制需要6个病毒编码的复制蛋白(BALF5、BMRF1、BALF2、BBLF4、BSLF1和BBLF2/3)。此外，即时早期反式激活蛋白BZLF1对oriLyt复制是必不可少的，即使其他病毒复制蛋白是由结构性活性启动子表达的。尽管单纯疱疹病毒来源结合蛋白UL9的功能等价物尚未被确定为EBV，但EBV oriLyt中只有两个小区域(“上游”和“下游”必需区域)是复制所绝对需要的。BZLF1与上游必需结构域的结合是oriLyt复制所必需的。我们最近发现，下游的必要结构域被BMRF1蛋白(病毒聚合酶处理因子)转录激活，并表明oriLyt上的小突变取消了BMRF1诱导的反式激活，也取消了oriLyt的复制。因此，BMRF1蛋白在oriLyt复制中似乎有两种不同的作用，既是聚合酶处理因子，又是转录激活因子。在这项资助中，我们建议研究病毒蛋白BMRF1和BZLF1对oriLyt的调控。我们的具体目标是：L)确定BMRF1转录激活oriLyt的机制，2)确定BMRF1的转录激活功能(除聚合酶处理功能外)是否是oriLyt复制所必需的，3)用电子显微镜检测BZLF1和BMRF1对oriLyt结构的影响，以及4)确定EB病毒(像其他疱疹病毒一样)在裂解感染过程中是否破坏与PML相关的核体，如果是，是否需要这种作用。我们假设BMRF1对oriLyt的转录激活是EBV裂解复制所必需的，BZLF1和/或BMRF1是oriLyt的功能性来源结合蛋白。