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

 描述(申请人提供)：所有基于脱氧核糖核酸的微生物，包括单纯疱疹病毒-1(HSV-1)，表现出突变频率约为0.003个突变/复制周期。有几个过程有助于保持这一水平的保真度，包括复制DNA聚合酶的核苷酸选择性、校对外切酶活性、复制后修复以纠正错误结合事件，以及其他DNA修复过程，如碱基切除修复。总体而言，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，基因组长约152kbp，编码约75个已知基因。HSV-1在人群中非常普遍，高达90%的美国人口对该病毒的血清检测呈阳性。通常情况下，病毒在上皮细胞中经历一个有效的裂解复制周期，其特征是病毒基因表达、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持续存在，在此期间没有病毒DNA复制，基因表达非常有限，也没有病毒产生。活动性HSV-1感染通常以口腔损害为特征。此外，疱疹角膜炎是导致失明和病毒性脑炎的主要原因，虽然不常见，但总是致命的。HSV-1的流行及其导致不同程度发病率和死亡率的反复感染的能力使其成为一个重大的公共卫生问题。我们以前发现了一个与HSV-1 DNA聚合酶(UL30蛋白)相关的新的5‘DRP和脱嘌呤/脱嘧啶裂解酶活性，报道了它与病毒尿嘧啶DNA糖基酶(UL2蛋白)的相互作用，并利用这些因子在体外重组了尿嘧啶启动的碱基切除修复。我们推测，涉及UL30和UL2的HSV-1碱基切除修复途径对病毒基因组的维持至关重要，发挥着关键的抗突变作用。具体地说，在这项建议中，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。具体工作如下：1)UL30裂解酶活性位点的鉴定和诱变。2)裂解酶缺陷UL30的生化特性，以确保其在其他方面具有功能。3)裂解酶缺陷病毒突变体的构建。4)裂解酶缺陷型HSV-1在细胞培养中复制效率和突变频率的测定。这一方法将使我们能够直接解决我们的假设，即UL30裂解酶活性具有抗突变功能，预测裂解酶缺陷的HSV-1表现出突变表型。证明UL30裂解酶的活性发挥了抗突变作用，这将为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。靶向UL30的DNA裂解酶活性可导致高度突变，从而导致在裂解复制期间和/或在潜伏病毒重新激活时产生“不适合”的病毒，可用于潜在的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.