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菌， 导致广泛的发病率和死亡率。这是已知的最常见的 美国的先天性病毒感染，有时严重 或致命后果；据估计，每年有3000-4000名婴儿 出生时有症状感染，并患有耳聋、失明、精神障碍 智障，或者死亡。免疫功能低下的人巨细胞病毒感染 否则重新激活可能是致命的。人巨细胞病毒是艾滋病的一个特殊问题 这可能是HIV致病的一个辅助因素。像其他疱疹一样 与病毒不同，CMV有裂解期和潜伏期。一种对 人巨细胞病毒DNA复制及其调控的分子机制 需要帮助制定有效的抗病毒战略。裂解相 DNA复制需要两个反式作用因素，如病毒- 特定的DNA聚合酶和最近发现的顺式作用序列， DNA复制的起源，oriLyt。拟议中的实验将 确定执行DNA复制的一整套病毒基因， 然后使用生物化学和免疫学来表征新的因素 方法：研究方法。其具体目的是：(I)定义最小子集 人巨细胞病毒溶血期DNA复制所必需和充分的基因； (Ii)表达已定义的基因，并提纯基因产物；及。(Iii) 用生化和免疫学方法对纯化后的产物进行表征 蛋白质。将使用的方法包括：(I)瞬时转染法- 互补分析以定位复制所需的新基因，以及 确认HSV 1型复制的候选HCMV同源物的作用 基因；(2)蛋白质表达的原核和真核系统； 以及(Iii)各种生化技术，包括电泳法， 柱层析和亲和层析，以纯化和鉴定 蛋白质因素。我们的长期目标是为全面的 对其分子机制和生物学策略的理解 人巨细胞病毒DNA复制和体外DNA的发育 复制系统。

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; by one estimate 3000-4000 infants annually are born with symptomatic infection and suffer deafness, blindness, mental retardation, or death. 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. Like other herpes viruses, CMV exhibits both lytic and latent phases. An 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 trans-acting factors such as the virus- specified DNA polymerase, and a recently identified cis-acting sequence, the origin of DNA replication, oriLyt. The experiments proposed will identify the complete set of virus genes that carry out DNA replication, then characterize novel factors using biochemical and immunological methods. The specific aims are: (i) to define the minimal subset of HCMV genes necessary and sufficient for HCMV lyti-phase DNA replication; (ii) to express the defined genes and purify the gene products; and (iii) to use biochemical and immunological methods to characterize the purified proteins. The methods to be used include: (i) a transient transfection- complementation assay to map novel genes required for replication,a nd to confirm the role of candidate HCMV homologs of HSV type 1 replication genes; (ii) prokaryotic and eukaryotic systems for protein expression; and (iii) various biochemical techniques including electrophoresis, column and affinity chromatography, to purify and characterize the protein factors. The long term goal is 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.