Molecular Genetics of HSV DNA Polymerase Gene

HSV DNA聚合酶基因的分子遗传学

• 批准号: 6623548
• 负责人: DONALD M COEN
• 金额: $ 38.7万
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623548
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; Herpesviridae disease; X ray crystallography; clinical research; cytomegalovirus; drug resistance; ganciclovir; gene mutation; genetic translation; herpes simplex virus 1; human subject; male; molecular genetics; protein protein interaction; protein structure function; virus DNA; virus genetics; virus protein; virus replication

DESCRIPTION (provided by applicant): 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, especially those caused by human cytomegalovirus (CMV) in immunosuppressed patients such as those with AIDS. 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 the interaction of the accessory subunit, HSV UL42, with DNA that permits sliding despite high affinity binding and the implications of this interaction for processive DNA synthesis. The UL42-DNA interaction will be explored using mutational approaches, protein-DNA crosslinking, measurements of affinity, on, off, and sliding rates, and single molecule studies and will be correlated with effects on processivity. Specific aim 3 is to determine mechanisms by which mutant CMV Pols, especially those altered in exonuclease motifs, resist ganciclovir (GCV) action, by analyzinf Pol interactions with GCV-TP and GCV-containing DNA. Specific aim 4 is to investigate CMV Pol's interaction with its accessory subunit, UL44. Interacting residues will be defined genetically and tested for their importance in CMV DNA and viral replication to determine if this interaction is a valid drug target. If so, a high throughput screening assay will be used to discover new antiviral drugs. Specific aim 5 is to use X-ray crystallography and, if necessary, nuclear magnetic resonance, to solve the three dimensional structures of domains of HSV US11, and HSV and CMV DNA polymerase and/or domains thereof to gain basic information regarding polymerase functions and as a starting point for drug discovery.

描述（由申请人提供）：本研究的长期目标 是对疱疹病毒DNA聚合酶的详细了解。这些酶， 其包括催化亚单元（Pol）和辅助亚单元，辅助亚单元 刺激长链DNA合成，是原型α-样DNA聚合酶 也是抗病毒药物的绝佳靶点治疗需要新药 疱疹病毒感染，特别是由人类巨细胞病毒引起的感染 (CMV)免疫抑制的患者，如艾滋病患者。具体目标1是 为了确定调节HSV Pol翻译的机制， 病毒的重要性。突变、RNA结合和翻译分析 将测试病毒体蛋白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 辅助亚单位，HSV UL 42，尽管高，但DNA允许滑动 亲和结合和这种相互作用对进行性DNA的影响 合成.将使用突变的方法探索UL 42-DNA相互作用。 方法，蛋白质-DNA交联，亲和力的测量，开，关， 滑动速率和单分子研究，并将与影响 关于持续性。具体目标3是确定突变体CMV Pol，特别是那些在核酸外切酶基序中改变的Pol，对更昔洛韦（GCV）具有抗性 通过分析Pol与GCV-TP和含GCV的DNA的相互作用， 具体目标4是研究CMV Pol与其附件的相互作用 亚基，UL 44。相互作用的残留物将在遗传学上进行定义，并检测 它们在CMV DNA和病毒复制中的重要性，以确定 相互作用是有效的药物靶点。如果是这样，高通量筛选试验 将用于发现新的抗病毒药物。具体目标5是使用X射线 晶体学，如果必要的话，核磁共振，来解决这个问题。 HSV US 11、HSV和CMV DNA的结构域的三维结构 聚合酶和/或其结构域，以获得关于 聚合酶的功能，并作为药物发现的起点。