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（由申请人提供）：疱疹病毒具有保守的复制机制，可以为抗病毒疗法提供潜在的靶标。为治疗目的进行了广泛的研究，已经对单纯疱疹病毒-1 DNA聚合酶（HSV POL）的催化亚基进行了广泛的研究，但其功能结构域和分子相互作用的完整补体尚未充分表征。 HSV POL的最近阐明的晶体结构揭示了未知功能的NH2-和NH2-末端域的存在。我们的最终目标是在功能上表征和评估此类域对酶促功能和病毒复制的重要性。具体而言，我们将首先确定1）如果NH2-末端结构域对于病毒DNA合成至关重要，并且2）如果NH2-末端结构域编码5'-3'RNase H活性，并且该域对于病毒复制至关重要。借助结构，我们将使用定向诱变的位点对工程师突变蛋白构建体，这些构建体可以通过体外表达系统表达，以评估聚合活性。 Vye使用细菌性人造染色体（BAC）技术，随后将构建突变体pel病毒，以评估在感染背景下引入突变的重要性。对于复制缺陷性突变病毒，我们将使用定量PCR测量HSV特异性基因的相对量，以确定是否损害了病毒DNA合成。进一步研究被感染细胞中突变pol的分子相互作用和酶促活性，并使用标准的生化技术可以实现纯化的蛋白质。这些研究将在病毒复制过程中进一步了解HSV POL功能，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。目前可用的治疗因素不足以容易发生耐药性感染的免疫功能低下的患者。对病毒复制过程中HSV-1 DNA聚合酶功能的增强理解可能为开发更有效的治疗提供了基础。