STRUCTURE AND FUNCTION OF THE HSV DNA POLYMERASE

HSV DNA 聚合酶的结构和功能

• 批准号: 3455439
• 负责人: DAVID I DORSKY
• 金额: $ 12.15万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455439
• 关键词: Alphaherpesvirinae; DNA binding protein; DNA directed DNA polymerase; DNA replication; Epstein Barr virus; Herpes simplex disease; acyclovir; affinity chromatography; antibacterial antibody; antiviral agents; chimeric proteins; chromosome complement; crystallization; enzyme inhibitors; enzyme mechanism; enzyme structure; gene deletion mutation; gene expression; gene mutation; genetic transcription; genetic translation; immunofluorescence technique; insect virus; laboratory rabbit; molecular cloning; nucleic acid sequence; oligonucleotides; reticulocytes; site directed mutagenesis; synthetic peptide; temperature sensitive mutant; tissue /cell culture; transfection; transposon /insertion element; virus DNA; virus genetics; virus replication; yeasts

The herpesvirus group (HSV 1 and 2, VZV, CMV, and EBV) cause serious infections in both normal and immunocompromised hosts. Recently, several inhibitors of viral DNA polymerases have emerged as clinically useful drugs for the treatment of some herpesvirus infections. The DNA polymerases of all the herpesviruses are quite similar but significant differences between them may have important implications for the development of more effective antiviral agents. The HSV DNA polymerase (pol) is an important model for the study of viral DNA replication and the mechanism of antiviral drugs. It has been well-characterized as an enzyme, the gene has been cloned and sequenced, and numerous genetic markers (temperature-sensitive and drug-resistance) have been defined. Recently, the pol gene has been expressed in a heterologous system as a functional product, which permits genetic manipulations of the pol gene to answer important questions about domain structure within pol and its interaction with antiviral agents. The broad, long-term objective of this study is to obtain a detailed understanding of the pol structure, mechanism, and interactions with inhibitors, leading to the rational design of new antiviral agents, possibly active against other homologous viral polymerases. The pol gene is biologically active when expressed in COS-1 cells; it is active enzymatically when expressed in yeast cells and also by in vitro translation in reticulocyte lysate. The effects of mutations introduced into the cloned pol gene can be tested by studying their effects on pol expressed in these heterologous system and by viruses expressing the mutant pol genes. Deletion analysis of the pol gene using the in vitro transcription- translation system will be carried out to define the minimum size of the polypeptide possessing core polymerase activity. This information can be used to express a deleted pol in bacteria to obtain a small enough molecule in large enough quantities to permit crystallization and detailed structural analysis. At least six domains within the polypeptide are conserved among the three prokaryotic and seven eukaryotic structures defining this class. Site-directed oligonucleotide mutagenesis will be carried out in these regions to define the contributions of these regions to enzymatic activity and sensitivity to inhibitors. Specific interaction of the in vitro translated pol with the 65 kD polymerase accessory protein will also be investigated with the pol mutants. The in vitro translated pol and mutants will also be used as targets for affinity-labeling by photoactive nucleotide analogs and specifically designed small DNA fragments.

疱疹病毒组（HSV 1 和 2、VZV、CMV 和 EBV）引起 正常和免疫功能低下宿主的严重感染。 最近，出现了几种病毒DNA聚合酶抑制剂 作为治疗某些疱疹病毒的临床有用药物 感染。 所有疱疹病毒的 DNA 聚合酶都相当 它们之间相似但显着的差异可能具有重要意义 对开发更有效的抗病毒药物的影响 代理。 HSV DNA 聚合酶 (pol) 是一个重要的模型 病毒DNA复制及抗病毒机制研究 药物。 它已被充分表征为一种酶，该基因具有 已被克隆和测序，以及许多遗传标记 （温度敏感和耐药性）已被定义。 最近，pol基因已在异源系统中表达 作为一种功能性产品，允许对 pol 基因回答有关结构域结构的重要问题 pol 内及其与抗病毒药物的相互作用。 广阔的、 本研究的长期目标是获得详细的 了解 pol 结构、机制和相互作用 与抑制剂，导致新的抗病毒药物的合理设计 剂，可能对其他同源病毒聚合酶具有活性。 pol基因在COS-1细胞中表达时具有生物活性； 当在酵母细胞中表达时，它具有酶活性，并且 通过网织红细胞裂解物的体外翻译。 的影响 引入克隆 pol 基因的突变可以通过以下方式进行测试 研究它们对这些异源基因中表达的 pol 的影响 系统和表达突变pol基因的病毒。 删除 使用体外转录分析 pol 基因 翻译系统将进行定义最小尺寸 具有核心聚合酶活性的多肽。 这 信息可用于表达细菌中删除的 pol 获得足够小、数量足够大的分子，以允许 结晶和详细结构分析。 至少六个 多肽内的结构域在三者之间是保守的 定义该类的原核和七种真核结构。 定点寡核苷酸诱变将在 这些区域来定义这些区域对 酶活性和对抑制剂的敏感性。 具体的 体外翻译的 pol 与 65 kD 的相互作用 聚合酶辅助蛋白也将用 pol 进行研究 突变体。 体外翻译的 pol 和突变体也将被使用 作为光敏核苷酸类似物亲和标记的靶标 以及专门设计的小DNA片段。