MOLECULAR GENETICS OF HSV DNA POLYMERASE GENE

这项研究的长期目标是详细了解 疱疹病毒DNA聚合酶。这些酶，包括一种催化 亚基(POL)和刺激长链DNA的辅助亚基 合成，是原型类阿尔法DNA聚合酶和优秀的 抗病毒药物的靶标。需要新药来治疗这种疾病 艾滋病患者中的疱疹病毒感染，特别是那些由 由人类巨细胞病毒(CMV)感染。具体目标1是确定 HSV POL翻译的调控机制及其对人类免疫功能的影响 病毒。突变、RNA结合和翻译分析将 检验病毒粒子蛋白US11刺激POL的假设 翻译早在感染之初，翻译低效在后 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 它的辅助亚单位UL42尽管稳定，但增强了加工性 UL42-DNA结合。突变对C末端DNA结合的影响 结构域将与POL活性和病毒的影响相关 复制。UL42机制将用有限的蛋白质分解来检验 和DNA足迹。具体目标3是通过以下方式确定机制 通过分析POL，哪些CMV突变体POL抵抗更昔洛韦(GCV)的作用 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UL44的相互作用。 相互作用的残基将从基因上定义并进行测试 在CMV DNA复制中的重要性。此信息将用作 发现抗病毒药物的起点。具体目标5是 求解HSV POL和UL42结构域的三维结构 和CMV Pol和UL44使用X射线结晶学获得基本 关于聚合酶功能的信息，并作为 药物发现。

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.