STRUCTURE & FUNCTION OF A CMV ORIGIN OF DNA REPLICATION

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 出生时有症状感染并患有耳聋、失明、精神 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 疱疹病毒CMV表现出裂解期和潜伏期。 一个 了解HCMV DNA复制的分子机制， 需要对它们进行调节以帮助开发有效的抗病毒药物， 战略布局 裂解期DNA复制需要反式作用 病毒特异性DNA聚合酶等因素，以及最近 确定了顺式作用序列，DNA复制起点，oriLyt。 提出的实验将阐明oriLyt的结构，研究 它在DNA复制中作用，并研究它与其他 DNA复制机制的组成部分。 具体目标是：（一） 确定CMV oriLyt的边界、结构和环境; ㈡ 以确定在已建立的边界内的哪些序列是 所需的，或有助于。（三）确定和 描述反式作用因子，这些因子可能与 oriLyt;和（iv）研究oriLyt功能的机制。的方法 （i）使用的方法包括：（i）构建广泛的突变系列 以及重组克隆中的缺失，然后将其功能性地 在用于定位oriLyt的瞬时测定中测试;（ii） 谱带转移和足迹研究， 位点特异性DNA-蛋白质相互作用，然后是柱和亲和性 （iii）用于纯化蛋白质因子的各种生物化学方法; 检测DNA结构、功能和构象的方法 在OriLyt中的片段。 长期目标是促进 全面了解分子机制和生物学 HCMV DNA复制的策略，以及体外 DNA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 in HIV pathogenesis. Like other herpesviruses 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 elucidate the structure of oriLyt, study its role in DNA replication, and examine its interaction with other elements of the DNA replication machinery. The specific aims are: (i) to establish the boundaries, structure and environment of CMV oriLyt; (ii) to determine-which sequences within the established boundaries are required for, or contribute to,. origin function; (iii) to identify and characterize trans-acting factors which may interact specifically with oriLyt; and (iv) to study the mechanisms of oriLyt function., The methods to be used include: (i) the construction of extensive series of mutations and deletions in recombinant clones which will then be functionally tested in the transient assay that was used to locate oriLyt; (ii) band-shifting and footprinting studies to identify and characterize site-specific DNA-protein interactions, followed by column and affinity chromatography to purify the protein factors; (iii) various biochemical methods to examine the DNA structure, function, and conformation of DNA segments within oriLyt. 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.