Molecular Genetics of HSV DNA Polymerase Gene

描述(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， 其包括催化亚单位(POL)和辅助亚单位 刺激长链DNA合成，是原型α-样DNA聚合酶 也是抗病毒药物的极佳靶点。治疗需要新药 疱疹病毒感染，尤其是由人类巨细胞病毒引起的 (CMV)免疫抑制患者，如艾滋病患者。具体目标1是 确定调节HSV POL及其翻译的机制 对病毒的重要性。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 辅助亚单位，HSV UL42，带有DNA，即使高水平也可以滑动 亲和力结合及其对进行性DNA的影响 综合。UL42-DNA的相互作用将使用突变 方法，蛋白质-DNA交联，亲和力测量，开，关，和 滑动率和单分子研究，并将与影响相关 关于过程性。具体目标3是确定突变的CMV POL，特别是那些核酸外切酶基序改变的POL，可以抵抗更昔洛韦(GCV) 作用，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，UL44。相互作用的残基将在基因上定义并进行测试 它们在CMV DNA和病毒复制中的重要性以确定这是否 相互作用是一个有效的药物靶点。如果是这样的话，一种高通量的筛选试验 将被用来发现新的抗病毒药物。具体目标5是使用X光 结晶学，如果必要的话，还有核磁共振，以解决 单纯疱疹病毒US11、单纯疱疹病毒和巨细胞病毒DNA结构域的三维结构 聚合酶和/或其结构域，以获得关于 聚合酶起作用，并作为药物发现的起点。

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.