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， 导致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 或致命的后果;据估计，每年有3000-4000名婴儿 出生时就有症状感染并患有耳聋、失明、精神疾病 迟缓或死亡 免疫功能低下者HCMV感染 否则重新激活可能是致命的 HCMV是艾滋病的一个特殊问题 患者，并可能是艾滋病毒发病机制的辅助因子。 像其他疱疹一样 病毒，CMV表现出裂解期和潜伏期。 了解 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复制需要两种反式作用因子，如病毒- 特定的DNA聚合酶，和最近鉴定的顺式作用序列， DNA复制起点oriLyt 提出的实验将 鉴定进行DNA复制的病毒基因的完整集合， 然后用生物化学和免疫学方法来表征新的因子， 方法. 具体目标是：（i）定义 HCMV裂解期DNA复制所必需的基因; (ii)表达所述确定的基因并纯化所述基因产物;以及（iii） 使用生物化学和免疫学方法来表征纯化的 proteins. 使用的方法包括：（i）瞬时转染- 互补试验，以绘制复制所需的新基因， 确认HSV 1型复制的候选HCMV同源物的作用 （ii）用于蛋白质表达的原核和真核系统; 和（iii）各种生物化学技术包括电泳， 柱和亲和层析，以纯化和表征 蛋白质因素 长期目标是促进全面的 了解的分子机制和生物学策略， HCMV DNA复制，并在体外DNA的发展 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.