HCMV DNA REPLICATION GENES IDENTIFIED BY TRANSIENT ASSAY

人类巨细胞病毒（HCMV）是一种常见的机会性病原体， 造成广泛的发病率和死亡率。 这是最常见的已知的 在美国先天性病毒感染，有时会出现严重的症状 或致命的后果；据估计，每年有 3000-4000 名婴儿 出生时患有症状感染并患有耳聋、失明、精神错乱 迟缓，或死亡。 在免疫功能低下的个体中，HCMV 感染 否则重新激活可能是致命的。 HCMV 是艾滋病中的一个特殊问题 患者，并且可能是 HIV 发病机制的辅助因子。 和其他疱疹一样 与病毒相比，CMV 表现出裂解期和潜伏期。 的理解 HCMV DNA 复制的分子机制及其调控是 需要帮助制定有效的抗病毒策略。 裂解期 DNA复制需要反式作用因子，例如病毒 特定的 DNA 聚合酶和最近鉴定的顺式作用序列， DNA复制的起点，oriLyt。 所提出的实验将 识别进行 DNA 复制的完整病毒基因组， 然后利用生化和免疫学来表征新因素 方法。 具体目标是： (i) 定义最小子集 HCMV 裂解期 DNA 复制所必需且充分的 HCMV 基因； (ii) 表达指定基因并纯化基因产物； (三) 使用生化和免疫学方法来表征纯化的 蛋白质。 使用的方法包括：（i）瞬时转染- 互补分析以绘制复制所需的新基因，以及 确认 1 型 HSV 复制的候选 HCMV 同源物的作用 基因； (ii) 用于蛋白质表达的原核和真核系统； (iii) 各种生化技术，包括电泳， 柱和亲和层析，以纯化和表征 蛋白质因素。 长期目标是为全面的 了解分子机制和生物学策略 HCMV DNA 复制，以及体外 DNA 的开发 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.