MOLECULAR GENETICS OF HSV DNA POLYMERASE GENE

HSV DNA 聚合酶基因的分子遗传学

• 批准号: 2061038
• 负责人: DONALD M COEN
• 金额: $ 25.23万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061038
• 关键词: Alphaherpesvirinae; Baculoviridae; DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; Herpes simplex disease; X ray crystallography; acyclovir; aminoacid; antiAIDS agent; antineoplastics; antiviral agents; biological signal transduction; cytomegalovirus; drug design /synthesis /production; drug resistance; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; eukaryote; exonuclease; gene expression; genetic manipulation; genetic mapping; genetic translation; molecular cloning; molecular genetics; mutant; nucleic acid sequence; pancreatic ribonuclease; protein sequence; radiotracer; site directed mutagenesis; virus DNA; virus genetics; virus replication

We wish to gain a comprehensive understanding of the regulation and function of the herpes simplex virus (HSV) DNA polymerase (pol) gene. HSV Pol is both an excellent model for eukaryotic replicative polymerases and an important target for antiviral drugs. HSV and other herpesviruses are important pathogens, especially in immunocompromised individuals such as AIDS patients and transplant and chemotherapy recipients. Drug resistance, due in part to pol mutations, is a growing problem. The mechanisms regulating the translational shut-off of pol gene expression and its importance to the virus will be analyzed through mutations that fail to down-regulate translation of pol mRNA. Studies of Pol function are facilitated by the availability of quantities of purified Pol from recombinant baculovirus-infected insect cells. The enzyme will be proteolytically cleaved into domains, which will be tested for various Pol activities. The structure of Pol and its domains will be examined by X-ray crystallography. Pol interactions with nucleic acids will be studied with spectroscopic, mobility shift and footprinting methods. Amino acids that bind deoxynucleoside substrates will be identified by affinity labeling. Genetic correlates to protein domains will be developed by analysis of mutations that exhibit intragenic complementation. The roles of conserved regions of Pol that are altered by acyclovir-resistance mutations will be investigated to determine which steps in the mechanism of acyclovir-triphosphate inhibition of Pol each mutation blocks. Amino acids hypothesized to be within active sites for the 3'-5' exonuclease and RNase H/5'3' exonuclease will be altered by site-specific mutagenesis and the importance of these residues for various Pol enzyme activities and for virus growth will be tested. A signal required for the localization of Pol to prereplicative sites in HSV-infected cells, which is dependent upon the viral single-stranded DNA binding protein, ICP8, will be sought by studies of mutations that prevent localization. The importance to the virus of this signal and of a domain that binds to the polymerase accessory protein, UL42, will be assessed by construction of HSV mutants. These studies should permit a detailed picture of the regulation and functional domains of Pol both in vitro and in infected cells and may uncover novel features of polymerases and new strategies for antiviral drug development.

我们希望对监管和 单纯疱疹病毒（HSV）DNA聚合酶（POL）基因的功能。 HSV POL既是真核复制性聚合酶的绝佳模型 以及抗病毒药物的重要靶标。 HSV和其他疱疹病毒 是重要的病原体，尤其是在免疫功能低下的个体中 作为艾滋病患者，移植和化学疗法接受者。 药品 抗性部分归因于POL突变，这是一个日益严重的问题。 调节POL基因的翻译关闭的机制 表达及其对病毒的重要性将通过 未能下调POL mRNA翻译的突变。 研究 POL功能可用于可用的数量 从重组杆状病毒感染的昆虫细胞中纯化的POR。 这 酶将蛋白水解裂解到域，将进行测试 用于各种POL活动。 POL及其域的结构将是 通过X射线晶体学检查。 pol与核酸相互作用 将对光谱，移动性转移和足迹进行研究 方法。 结合脱氧核苷底物的氨基酸将是 通过亲和力标签确定。 遗传与蛋白质结构域相关 将通过分析表现出内部性的突变的分析来开发 互补。 改变的POL保守区域的作用 通过抗螺旋藻的抗性突变将进行研究，以确定哪个 阿西克洛维尔 - 三磷酸抑制POL的机制的步骤 突变块。 氨基酸假设是在活跃位点的 3'-5'外切核酸酶和RNase H/5'3'外丝丝将通过 位点特异性诱变及这些残基的重要性 将测试各种pol酶活性和病毒生长。 一个 将POL定位到预核位点所需的信号 HSV感染的细胞，该细胞取决于病毒单链DNA 结合蛋白ICP8将通过对突变的研究寻求 防止本地化。 对该信号和该信号病毒的重要性 与聚合酶辅助蛋白UL42结合的域将是 通过构建HSV突变体评估。 这些研究应允许 POL的调节和功能域的详细图片 体外和受感染的细胞，可能会发现聚合酶的新特征 以及抗病毒药物开发的新策略。