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

 描述（由申请方提供）：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。几种过程有助于维持这种保真度水平，包括复制型DNA聚合酶的核苷酸选择性、校正外切核酸酶活性、复制后修复以纠正错误掺入事件，以及其他DNA修复过程如碱基切除修复。总的来说，我们感兴趣的是HSV-1基因组复制和维持的机制，它们在病毒生物学中的作用以及它们作为抗病毒治疗靶点的潜力。HSV-1是一种大的双链DNA病毒，基因组约为152 kbp，编码约75个已知基因。HSV-1在人群中非常广泛，高达90%的美国人口对该病毒检测呈血清阳性。通常，病毒在上皮细胞中经历生产性裂解复制周期，其特征在于病毒基因表达、病毒DNA复制和新病毒产生。HSV-1通过在感觉神经元中建立潜伏期而在其宿主中持续存在，在此期间没有病毒DNA复制，非常有限的基因表达并且没有病毒产生。活动性HSV-1感染通常以口腔病变为特征。此外，疱疹性角膜炎是失明的主要原因，病毒性脑炎虽然不常见，但总是致命的。HSV-1的流行及其引起具有不同程度发病率和死亡率的复发性感染的能力使其成为重要的公共卫生问题。我们先前鉴定了一种新的与HSV-1 DNA聚合酶（UL 30蛋白）相关的5' dRP和脱嘌呤/脱嘧啶裂解酶活性，报道了其与病毒尿嘧啶DNA糖基化酶（UL 2蛋白）的相互作用，并利用这些因子在体外重建尿嘧啶启动的碱基切除修复。我们推测，HSV-1碱基切除修复途径涉及UL 30和UL 2是至关重要的病毒基因组的维护，执行一个关键的抗突变的作用。具体而言，在本提案中，我们试图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。这将如下完成：1）UL 30裂解酶活性位点的鉴定和诱变。2)裂解酶缺陷型UL 30的生物化学表征，以确保其具有其他功能。3)裂解酶缺陷型病毒突变体的构建。4)测定细胞培养物中裂解酶缺陷型HSV-1的复制效率和突变频率。这种方法将使我们能够直接解决我们的假设，即UL 30裂解酶活性实现抗增变因子功能，即裂解酶缺陷型HSV-1表现出增变因子表型的预测。证明UL 30的裂解酶活性执行抗突变作用将为DNA修复过程在HSV-1基因组维持中的重要性提供重要的新见解。靶向UL 30的DNA裂解酶活性以引起超诱变，从而导致在裂解复制期间和/或在潜伏病毒再活化时产生“不适合”病毒，可用于潜在drg开发。

 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.