MOLECULAR GENETICS OF HSV DNA POLYMERASE GENE

HSV DNA 聚合酶基因的分子遗传学

• 批准号: 2003269
• 负责人: DONALD M COEN
• 金额: $ 28.31万
• 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 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2003269
• 关键词: Alphaherpesvirinae; DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; X ray crystallography; cytomegalovirus; drug resistance; enzyme activity; ganciclovir; gene mutation; genetic translation; messenger RNA; molecular cloning; protein structure function; virus DNA; virus genetics; virus protein; virus replication

The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are prototype alpha-like DNA polymerases and excellent targets for antiviral drugs. New drugs are needed for treatment of herpesvirus infections in patients with AIDS, especially those caused by human cytomegalovirus (CMV). Specific aim 1 is to determine mechanisms that regulate translation of HSV Pol and their importance to the virus. Mutational, RNA-binding, and translational analyses will test the hypotheses that a virion protein, US11, stimulates Pol translation early in infection, while inefficient translation later is beneficial to the virus. Specific aim 2 is to analyze functions of the small C-terminal domain of HSV Pol and to determine the mechanism by which the accessory subunit, UL42, enhances processivity despite stable UL42-DNA binding. Effects of mutations on DNA-binding by the C-terminal domain will be correlated with effects on Pol activity and virus replication. UL42 mechanisms will be examined using limited proteolysis and DNA footprinting. Specific aim 3 is to determine mechanisms by which mutant CMV Pols resist ganciclovir (GCV) action, by analyzing Pol interactions with GCV-TP and GCV-containing DNA. Specific aim 4 is to investigate CMV Pol's interaction with its accessory protein, UL44. Interacting residues will be defined genetically and tested for their importance in CMV DNA replication. This information will serve as a starting point for discovery of antiviral drugs. Specific aim 5 is to solve the three dimensional structures of domains of HSV Pol and UL42 and CMV Pol and UL44 using x-ray crystallography to gain basic information regarding polymerase functions and as a starting point for drug discovery.

本研究的长期目标是详细了解 疱疹病毒DNA聚合酶。 这些酶，其中包括催化 亚基（Pol）和刺激长链DNA的辅助亚基 是原型α-样DNA聚合酶， 抗病毒药物的靶点。 需要新药来治疗 艾滋病患者的疱疹病毒感染，特别是那些 人类巨细胞病毒（CMV）。 具体目标1是确定 调节HSV Pol翻译的机制及其对 病毒 突变、RNA结合和翻译分析将 检验病毒体蛋白US 11刺激Pol 在感染的早期，翻译效率低下， 对病毒有益。 具体目标2是分析 HSV Pol的小C-末端结构域，并通过 其中辅助亚基UL 42增强了持续合成能力， UL 42-DNA结合。 突变对C-末端DNA结合的影响 结构域将与对Pol活性和病毒的影响相关 复制的 将使用有限的蛋白水解检查UL 42机制 和DNA足迹 具体目标3是确定机制， 突变CMV Pos抵抗更昔洛韦（GCV）的作用，通过分析Pos 与GCV-TP和含GCV的DNA的相互作用。 具体目标4是 研究CMV Pol与其辅助蛋白UL 44相互作用。 相互作用的残基将在遗传学上进行定义，并对其进行检测。 在CMV DNA复制中的重要性。 这些信息将作为 发现抗病毒药物的起点。 具体目标5是 解析HSV Pol和UL 42结构域的三维结构 和CMV Pol和UL 44使用X射线晶体学获得基本的 关于聚合酶功能的信息并作为起点 药物发现