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

描述(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点。单纯疱疹病毒1型DNA聚合酶(HSV Pol)的催化亚单位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的表征。最近阐明的HSV Pol的晶体结构揭示了NH2-和前NH2-末端结构域的存在，功能未知。我们的最终目标是从功能上描述和评估这些结构域对POL酶功能和病毒复制的意义。具体地说，我们将首先确定1)前NH2末端结构域是否对病毒DNA合成至关重要，2)NH2末端结构域是否编码5‘-3’RNaseH活性以及该结构域是否对病毒复制至关重要。借助于结构，我们将使用定点突变来设计突变蛋白结构，这些突变蛋白结构可以通过体外表达系统表达，以评估聚合活性。利用细菌人工染色体(BAC)技术，VYE随后将构建突变的PEL病毒，以评估引入的突变在感染背景下的意义。对于复制缺陷突变病毒，我们将使用定量PCR来测量HSV特异性基因的相对数量，以确定病毒DNA合成是否受到损害。进一步研究突变体Pol在感染细胞内和作为纯化蛋白的分子相互作用和酶活性，可以使用标准的生化技术来实现。这些研究将进一步加深我们目前对HSV Pol在病毒复制过程中的功能的理解，这可能为疾病的治疗提供基础，并为真核同源物提供一个模型系统。疱疹病毒是许多医学上相关疾病的原因，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免疫功能低下的患者，因为这些患者很容易出现耐药感染。进一步了解HSV-1DNA聚合酶在病毒复制过程中的功能可能为开发更有效的治疗方法提供基础。

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.