HCMV DNA REPLICATION GENES IDENTIFIED BY TRANSIENT ASSAY

人巨细胞病毒(HCMV)是一种常见的条件致病原，可引起 广泛的发病率和死亡率。这是已知的最常见的先天性 美国的病毒感染，有时严重或致命 后果。在免疫受损的个体中，HCMV感染或 重新激活可能是致命的。人巨细胞病毒在艾滋病患者中是一个特别的问题， 可能是HIV致病的一个辅助因素。更好地理解 人巨细胞病毒DNA复制的分子机制及其调控 需要帮助制定有效的抗病毒战略。裂解相 DNA复制需要DNA复制的顺式作用起点， OriLyt，以及诸如病毒特异性DNA等交易因子 聚合酶。11个不同的人巨细胞病毒基因座，加上oriLyt，与 通过瞬时互补实验实现原点介导的DNA复制 使用病毒基因组的亚克隆。此外，开放阅读框架 位于oriLyt内的UL59可能也是必需的。具体目标 这项建议的内容如下：(I)确定是否需要UL59来补充 DNA合成；(Ii)确定是否某些基因座是 在反式中所需的瞬时互补仅仅是为了 必需复制蛋白的表达，或者如果所有的基因座都编码 直接参与启动或执行DNA的蛋白质 复制；以及(Iii)过度表达、纯化和表征候选 复制蛋白UL84、UL36、UL37、UL37ex1和IRS1/TRS1，以及TO 准备免疫学和生化工具，用于详细分析它们的 在DNA合成中的可能作用。这些实验将特别集中于 这些蛋白质最有可能在调节和 促进印心。将使用的方法包括：(I)瞬变 利用异源启动子构建的互补分析 允许表达不依赖于HCMV指定的反式激活剂；(Ii) 蛋白质过表达的原核和真核系统；以及(Iii) 标准生化技术，包括电泳和柱层析 亲和层析纯化和鉴定蛋白质因子。 长期目标是有助于全面了解 人巨细胞病毒DNA复制的分子机制和生物学策略 以及开发体外DNA复制系统，并帮助 新型抗病毒药物的开发。

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.