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: 7754567
• 负责人: SHARIYA L. TERRELL
• 金额: $ 3.22万
• 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/7754567
• 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; Simplexvirus DNA polymerase; 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（由申请方提供）：疱疹病毒表现出保守的复制机制，可为抗病毒治疗提供潜在靶点。单纯疱疹病毒1型DNA聚合酶（HSV Pol）的催化亚基已被广泛研究用于治疗目的，但其完整的功能结构域和分子相互作用尚未得到充分表征。最近阐明的HSV Pol的晶体结构揭示了未知功能的NH 2-和前NH 2-末端结构域的存在。我们的最终目标是在功能上表征和评估这些结构域对Pol酶功能和病毒复制的意义。具体而言，我们将开始确定1）前NH 2末端结构域是否是病毒DNA合成所必需的，和2）NH 2末端结构域是否编码5 '-3' RNA酶H活性，以及该结构域是否是病毒复制所必需的。借助于结构，我们将使用定点诱变来工程化突变蛋白质构建体，所述突变蛋白质构建体可以通过体外表达系统表达以用于评价聚合活性。使用细菌人工染色体（BAC）技术，vye随后将构建突变pel病毒，以评估在感染背景下引入突变的意义。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合成是否受损。可以使用标准生物化学技术来实现对感染细胞内的突变Pol和纯化蛋白质的分子相互作用和酶活性的进一步研究。这些研究将进一步加深我们目前对HSV Pol在病毒复制过程中功能的理解，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原因，包括口腔和生殖器疱疹，脑炎和癌症。目前可用的治疗方法不足以治疗易发生耐药性感染的免疫功能低下患者。对HSV-1 DNA聚合酶在病毒复制过程中功能的深入了解可能为开发更有效的治疗方法提供基础。