Functional Characterization of HSV-1 DNA Polymerase preN-and N -Terminal Domains

HSV-1 DNA 聚合酶前 N 端和 N 端结构域的功能表征

• 批准号: 8078092
• 负责人: SHARIYA L. TERRELL
• 金额: $ 2.9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078092
• 关键词: Address; Antiviral Therapy; Bacterial Artificial Chromosomes; Bacteriophages; Binding; Binding Sites; Biochemical; Biological Models; Catalytic Domain; Cells; Cessation of life; Clinical; Complement; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Development; Disease; Drug resistance; Engineering; Evaluation; Exhibits; Family; Foundations; Genes; Goals; Growth; Herpes encephalitis; Herpesviridae; Herpesvirus 1; Homologous Gene; Immunocompromised Host; In Vitro; Infection; Investigation; Kinetics; Location; Malignant Neoplasms; Measures; Metal Ion Binding; Mutation; N-terminal; Oral; Phosphodiesterase I; Polymerase; Process; Production; Proteins; RNA Binding; RNA Recognition Motif; RNA primers; Relative (related person); Replication-Associated Process; Reporting; Research Proposals; Ribonuclease H; Sequence Homology; Simplexvirus; Site; Site-Directed Mutagenesis; Structure; System; Techniques; Technology; Testing; Therapeutic Studies; Viral; Viral Genome; Viral Physiology; Virus; base; beta pleated sheet; effective therapy; genital herpes; member; mutant; polymerization; research study; spleen exonuclease; viral DNA

DESCRIPTION (provided by applicant): Herpesviruses exhibit conserved replication mechanisms that can provide potential targets for antiviral therapies. The catalytic subunit of Herpes simplex virus-1 DNA Polymerase (HSV Pol) has been extensively studied for therapeutic purposes, yet its full complement of functional domains and molecular interactions has yet to sufficiently characterized. The recently elucidated crystal structure of HSV Pol revealed the presence of NH2- and pre-NH2- terminal domains of unknown function. Our ultimate goal is to functionally characterize and evaluate the significance of such domains to Pol enzymatic functions and viral replication. Specifically, we will begin by determining 1) if the pre-NH2-terminal domain is essential for viral DNA synthesis and 2) if the NH2-terminal domain encodes 5'-3' RNase H activity and if this domain is essential for viral replication. With the aid of structure, we will use site directed mutagenesis to engineer mutant protein constructs that can be expressed via in vitro expression systems for evaluation of polymerization activity. Using bacterial artificial chromosome (BAC) technology, vye will subsequently construct mutant pel viruses in order to evaluate the significance of introduced mutations within the context of infection. For replication-defective mutant viruses, we will measure the relative amounts of an HSV-specific gene using quantitative PCR to determine if viral DNA synthesis has been impaired. Further investigation into the molecular interactions and enzymatic activities of mutant Pol within infected cells and as purified protein can be achieved using standard biochemical techniques. These studies will further our present understanding of HSV Pol function within the process of viral replication which may provide the foundation for disease treatment and serve as a model system for eukaryotic homologues. Herpesviruses are the cause of many medically relevant conditions including oral and genital herpes, encephalitis, and cancer. Currently available treatments are inadequate for immunocompromised patients in whom drug resistant infections readily develop. An enhanced understanding of HSV-1 DNA Polymerase functions within viral replication processes may provide the basis for development of more effective treatments.

描述（由申请人提供）：疱疹病毒表现出保守的复制机制，可以为抗病毒治疗提供潜在的靶标。单纯疱疹病毒 1 DNA 聚合酶 (HSV Pol) 的催化亚基已被广泛研究用于治疗目的，但其完整的功能域和分子相互作用尚未得到充分表征。最近阐明的 HSV Pol 晶体结构揭示了功能未知的 NH2- 和前 NH2- 末端结构域的存在。我们的最终目标是功能表征和评估这些结构域对 Pol 酶功能和病毒复制的重要性。具体来说，我们将首先确定 1) 前 NH2 末端结构域是否对于病毒 DNA 合成至关重要，2) NH2 末端结构域是否编码 5'-3' RNase H 活性以及该结构域是否对于病毒复制至关重要。借助结构，我们将使用定点诱变来设计突变蛋白构建体，该构建体可以通过体外表达系统表达以评估聚合活性。使用细菌人工染色体（BAC）技术，vye 随后将构建突变体病毒，以评估感染背景下引入的突变的重要性。对于复制缺陷型突变病毒，我们将使用定量 PCR 测量 HSV 特异性基因的相对量，以确定病毒 DNA 合成是否受到损害。使用标准生化技术可以进一步研究受感染细胞内突变体 Pol 以及纯化蛋白质的分子相互作用和酶活性。这些研究将进一步加深我们对病毒复制过程中 HSV Pol 功能的了解，这可能为疾病治疗提供基础，并作为真核同源物的模型系统。疱疹病毒是许多医学相关疾病的病因，包括口腔和生殖器疱疹、脑炎和癌症。目前可用的治疗方法对于免疫功能低下的患者来说是不够的，因为这些患者容易产生耐药性感染。增强对病毒复制过程中 HSV-1 DNA 聚合酶功能的了解可能为开发更有效的治疗方法提供基础。