STRUCTURE & FUNCTION OF A CMV ORIGIN OF DNA REPLICATION

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，有时是严重的 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致病过程中的一个辅助因素。像其他人一样 疱疹病毒CMV有裂解期和潜伏期。一个 了解人巨细胞病毒DNA复制和转录的分子机制 需要对它们进行监管，以帮助开发有效的抗病毒药物 战略。裂解期DNA复制需要两种反式作用 例如病毒特有的DNA聚合酶，以及最近的 确定了顺式作用序列，DNA复制的起始点，oriLyt。 提出的实验将阐明oriLyt的结构，研究 它在DNA复制中的作用，并研究它与其他 DNA复制机制的要素。具体目标是：(I) 建立CMV oriLyt的边界、结构和环境；(Ii) 以确定已建立的边界内的哪些序列 需要的，或对…有贡献的。原产地函数；(Iii)识别和 描述可能与特定基因相互作用的反式作用因子 (4)研究oriLyt的作用机制，方法 要使用的方法包括：(1)构建广泛的突变序列 以及在重组克隆中的缺失，这些克隆将在功能上 在用于定位oriLyt的瞬时检测中进行测试；(Ii) 频带移动和足迹研究，以识别和表征 特定部位的DNA-蛋白质相互作用，然后是柱状和亲和力 蛋白质因子的层析纯化；(Iii)各种生化 方法检测DNA的结构、功能和构象 OriLyt内的线段。我们的长期目标是为 对分子机制和生物学的全面理解 人巨细胞病毒DNA复制策略及其在体外发育中的作用 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.