HCMV DNA REPLICATION GENES IDENTIFIED BY TRANSIENT ASSAY

人巨细胞病毒（HCMV）是一种常见的机会致病菌， 广泛的发病率和死亡率。这是最常见的已知先天性 病毒感染在美国，有时严重或致命 后果在免疫功能低下的个体中，HCMV感染或 重新激活可能是致命的。HCMV在艾滋病患者中是一个特别的问题， 可能是HIV致病的辅助因子。更好地理解 人巨细胞病毒DNA复制及其调控的分子机制， 需要帮助开发有效的抗病毒策略。裂解相 DNA复制需要DNA复制的顺式作用起点， oriLyt和反式作用因子，如病毒特异性DNA 聚合酶。11个不同的HCMV基因座，加上oriLyt，涉及 通过瞬时互补实验的起点介导的DNA复制 使用病毒基因组的亚克隆。此外，开放式阅读框架 也可能需要位于oriLyt中的UL 59。具体目标 （一）确定是否需要UL 59来补充 （二）确定是否存在某些基因座， 瞬时互补所需的反式， 表达必需的复制蛋白，或者如果所有基因座编码 直接参与启动或执行DNA的蛋白质 复制;和（iii）过表达、纯化和表征候选物 复制蛋白UL 84、UL 36、UL 37、UL 37 ex 1和IRS 1/TRS 1，以及 准备免疫学和生物化学工具，用于详细分析 DNA合成中的可能作用这些实验将特别关注 离子这些蛋白质，最有可能发挥作用， 促进启蒙。使用的方法包括：（i）瞬态 使用异源启动子构建体的互补测定，所述异源启动子构建体被设计成 允许独立于HCMV特异性反式激活因子的表达;（ii） 用于蛋白质过表达的原核和真核系统;和（iii） 标准生化技术，包括电泳和柱， 亲和层析纯化和表征蛋白因子。 长期目标是促进全面了解 HCMV DNA复制的分子机制和生物学策略 以及体外DNA复制系统的开发，并有助于 开发新的抗病毒药物。

Human cytomegalovirus (HCMV) is a frequent opportunistic pathogen, causing extensive morbidity and mortality. It is the most common known congenital virus infection in the United States, sometimes with serious or fatal consequences. In immunocompromised individuals HCMV infection or reactivation can be fatal. HCMV is a particular problem in AIDS patients, and may be a cofactor for HIV pathogenesis. A better understanding of the molecular mechanisms of HCMV DNA replication and their regulation is needed to aid in developing effective antiviral strategies. Lytic-phase DNA replication requires both a cis-acting origin of DNA replication, oriLyt, and transacting factors such as the virus-specified DNA polymerase. Eleven distinct HCMV loci, plus oriLyt, were implicated in origin-mediated DNA replication by transient complementation experiments using subclones of the viral genome. In addition, the open reading frame UL59, which lies within oriLyt, might also be required. The specific aims of this proposal are: (i) to determine if UL59 is required to complement DNA synthesis; (ii) to establish whether some of the loci that are required in trans for transient complementation are needed solely to allow expression of essential replication proteins, or if all loci encode proteins that participate directly in initiating or performing DNA replication; and (iii) to overexpress, purify, and characterize candidate replication proteins UL84, UL36, UL37, UL37ex1, and IRS1/TRS1, and to prepare immunological and biochemical tools for detailed analysis of their possible roles in DNA synthesis. These experiments will focus especially Ion those proteins that most likely play a role in regulating and promoting initiation. The methods to be used include: (i) a transient complementation assay using heterologous promoter constructs designed to allow expression independent of HCMV-specified transactivators; (ii) prokaryotic and eukaryotic systems for protein overexpression; and (iii) standard biochemical techniques including electrophoresis and column and affinity chromatography to purify and characterize the protein factors. The long term goals are to contribute to a comprehensive understanding of the molecular mechanisms and biological strategies of HCMV DNA replication and to the development of an in vitro DNA replication system, and to aid development of novel antiviral agents.