Role of herpes simplex virus polymerase DNA repair activity in viral replication

单纯疱疹病毒聚合酶 DNA 修复活性在病毒复制中的作用

基本信息

批准号：
9089885
负责人：
Paul E Boehmer
金额：
$ 7.68万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9089885
关键词：
5&apos -deoxyribose phosphate lyase Active Sites Address Afferent Neurons Antiviral Therapy Arginine Attenuated Baculoviruses Base Excision Repairs Biochemical Biological Assay Biology Blindness C-terminal Cell Culture Techniques Cell Nucleus Cells Cornea DNA DNA Repair DNA Repair Pathway DNA biosynthesis DNA-Directed DNA Polymerase Defect Development Double Stranded DNA Virus Encephalitis Ensure Epithelial Cells Event Excision Exhibits Exonuclease Family member Frequencies Gene Expression Genes Genome Goals Herpes Labialis Herpesviridae Herpesvirus 1 Human In Vitro Induced Mutation Infection Intervention Keratitis Knowledge Latent Virus Location Lyase Lysine Lytic Maintenance Microbe Morbidity - disease rate Mutagenesis Mutation Nucleotides Pain Pathway interactions Phenotype Phosphodiesterase I Play Polymerase Population Prevalence Process Production Property Proteins Public Health Reaction Recurrence Reporting Research Role Schiff Bases Simplexvirus Site Structure-Activity Relationship System Testing Uracil Viral Viral Encephalitis Viral Genes Viral Genome Virus Virus Replication Wit amino group base genome integrity improved insight interest lytic replication mortality mutant novel oral lesion pathogen public health relevance recombinational repair reconstitution repaired seropositive uracil-DNA glycosylase viral DNA

项目摘要

DESCRIPTION (provided by applicant): All DNA based microbes including herpes simplex virus-1 (HSV-1), exhibit mutation frequencies in the order of ~0.003 mutations/replication cycle. Several processes contribute towards maintaining this level of fidelity including the nucleotide selectivity of the replicative DNA polymerase, proofreading exonuclease activity, post- replication repair to correct mis-incorporation events, and other DNA repair processes such as base excision repair. Overall, we are interested in the mechanisms that underlie genome replication and maintenance in HSV-1, their roles in the biology of the virus and their potential as targets for anti-viral therapy. HSV-1 is a large double-strand DNA virus with a genome of ~152 kbp, encoding ~75 known genes. HSV-1 is extremely widespread in the population, with up to 90% of the US population testing seropositive for the virus. Typically, the virus undergoes a productive lytic replication cycle in epithelial cells, characterized by viral gene expression, vral DNA replication and new virus production. HSV-1 persists in its host by establishing latency in sensory neurons during which there is no viral DNA replication, very limited gene expression and no virus production. Active HSV-1 infections are commonly characterized by oral lesions. In addition, herpes keratitis is a leading cause of blindness and viral encephalitis, while uncommon, is invariably fatal. The prevalence of HSV-1 and its ability to cause recurrent infections with varying degrees of morbidity and mortality make it a significant public health problem. We previously identified a novel 5' dRP and apurinic/apyrimidinic lyase activity associated with the HSV-1 DNA polymerase (UL30 protein), reported its interaction with the viral uracil DNA glycosylase (UL2 protein) and reconstituted uracil-initiated base excision repair utilizing these factors in vitro. We hypothesize that the HSV-1 base excision repair pathway involving both UL30 and UL2 is critical to viral genome maintenance, performing a critical anti-mutator role. Specifically, in this proposal we seek to address the role of the lyase activity in viral replication and its impact on replication and mutation frequency. This will be accomplished as follows: 1) Identification and mutagenesis of the UL30 lyase active site. 2) Biochemical characterization of the lyase-deficient UL30 to ensure that it is otherwise functional. 3) Construction of a lyase-deficient viral mutant. 4) Determination of replication efficiency and mutation frequency of lyase-deficient HSV-1 in cell culture. This approach will allow us to directly address our hypothesis that the UL30 lyase activity fulfills an anti-mutator function, wit the prediction that lyase-deficient HSV-1 exhibits a mutator phenotype. Demonstrating that the lyase activity of UL30 performs an anti-mutator role will provide significant new insight into the importance of DNA repair processes in HSV-1 genome maintenance. Targeting the DNA lyase activity of UL30 to cause hypermutagenesis and thereby result in the production of "unfit" virus during lytic replication and/or upon reactivation of latent virus may be exploited for potential drg development.

描述（由申请人提供）：所有基于 DNA 的微生物，包括单纯疱疹病毒 1 (HSV-1)，均表现出约 0.003 个突变/复制周期的突变频率。有几个过程有助于维持这种保真度水平，包括核苷酸选择性。复制DNA聚合酶、校对核酸外切酶活性、复制后修复以纠正错误掺入事件以及其他DNA修复过程（例如碱基切除修复）总体而言，我们对其机制感兴趣。 HSV-1 基因组复制和维持的基础、它们在病毒生物学中的作用及其作为抗病毒治疗靶点的潜力。HSV-1 是一种大型双链 DNA 病毒，基因组约为 152 kbp，编码。 HSV-1 约有 75 个已知基因，在人群中极为广泛，高达 90% 的美国人检测出该病毒血清呈阳性。通常，该病毒在上皮细胞中经历高效的裂解复制周期，其特征是病毒基因。 HSV-1 通过在感觉神经元中建立潜伏期而持续存在于其宿主中，在此期间没有病毒 DNA 复制、非常有限的基因表达和没有病毒产生。此外，疱疹性角膜炎是导致失明和病毒性脑炎的主要原因，虽然不常见，但它总是致命的。HSV-1 的流行及其引起不同程度发病率和死亡率的反复感染的能力使其成为重要的公众疾病。我们之前发现了与 HSV-1 DNA 聚合酶（UL30 蛋白）相关的新型 5' dRP 和无嘌呤/无嘧啶裂合酶活性，报告了其与病毒尿嘧啶 DNA 糖基化酶（UL2 蛋白）的相互作用，并重建了尿嘧啶引发的碱基切除。我们率先发现涉及 UL30 和 UL2 的 HSV-1 碱基切除修复途径对于病毒基因组的维持至关重要，具体来说，在本提案中，我们寻求解决裂解酶活性在病毒复制中的作用及其对复制和突变频率的影响，这将通过以下方式完成：1）UL30的鉴定和诱变。 2) 裂解酶缺陷型 UL30 的生化特征，以确保其具有其他功能。 3) 构建裂解酶缺陷型病毒突变体。这种方法将使我们能够直接解决我们的假设，即 UL30 裂解酶活性实现抗突变功能，并预测裂解酶缺陷的 HSV-1 表现出突变表型。证明 UL30 裂解酶活性具有抗突变作用，将为了解 DNA 修复过程在 HSV-1 基因组维护中的重要性提供重要的新见解。 UL30引起超突变并由此导致在裂解复制期间和/或在潜伏病毒重新激活时产生“不合适”病毒，可用于潜在的药物开发。