Uracil DNA Glycosylases in Gammaherpesvirus Pathogenesis and B Cell Development

项目摘要 每种疱疹病毒编码哺乳动物尿嘧啶DNA糖基化酶（vUNG）的病毒同系物。哺乳动物UNG是 一种去除错误掺入和致突变尿嘧啶的酶，留下脱碱基位点，通常由 宿主碱基切除修复途径。虽然它们之间存在保守的序列和体外活性， vUNG和宿主UNG，关于vUNG在病毒生命周期中的功能或对宿主基因组的影响知之甚少 DNA修复研究表明，疱疹病毒vUNG可能与病毒DNA复制的组分相互作用 然而，其在原代细胞中的晚期复制事件中的作用是不确定的。我们利用鼠 γ疱疹病毒68（MHV68）作为人γ疱疹病毒、EB病毒和 卡波西肉瘤相关疱疹病毒我们的研究表明，疱疹病毒vUNG在 致病性，因为vUNG缺陷型病毒具有主要的复制和感染性缺陷。γ疱疹病毒感染 在B细胞中建立潜伏期，这一过程可能导致 永生化和B细胞转化。在GC B细胞中，宿主UNG活性过程AID诱导尿嘧啶， 触发抗体多样化，这一过程也会触发与以下疾病相关的染色体易位 淋巴瘤我们的研究表明，vUNG可以处理基因组DNA中的尿嘧啶，但修复结果不同 主持人吴vUNG抑制抗体突变和类别转换重组， 催化和非催化依赖性表明vUNG的支架功能。因此，vUNG有潜力 有助于改变病毒发病机制、免疫力和恶性肿瘤。我们提出研究来了解 vUNG促进病毒发病机制并破坏宿主抗体多样化。在目标1中，结构和 将研究vUNG对DNA损伤的无错修复的生物化学基础。差异 vUNG和宿主UNG将被表征以确定导致以下结果的结构域和相互作用伙伴： 无错误修复与易出错修复结果的差异。在目标2中，vUNG促进 将研究γ疱疹病毒的发病机理。vUNG酶功能的贡献和 将表征支架功能。在目标3中，γ疱疹病毒感染和vUNG对B的影响 将研究细胞发育。将在研究期间检查B细胞活化和免疫球蛋白库。 MHV 68感染。将在急性感染和潜伏期分析抗体库和自身反应性 重新激活我们的目标横跨分子免疫学和分子病毒学领域， γ疱疹病毒发病机制和B细胞生物学。拟议的研究应阐明机制， 该vUNG支持病毒复制、感染和免疫球蛋白多样化的病毒颠覆。

Project Summary Each herpesvirus encodes a viral homolog of mammalian uracil DNA glycosylase (vUNG). Mammalian UNG is an enzyme that removes misincorporated and mutagenic uracils, leaving an abasic site typically repaired by the host base excision repair pathways. Although there is conserved sequence and in vitro activity between vUNG and host UNG, little is known regarding vUNG function in the virus lifecycle or effect on host genome DNA repair. Studies indicate that herpesvirus vUNG may interact with components of the viral DNA replication machinery; however, its role in late stage replication events in primary cells is uncertain. We utilize murine gammaherpesvirus 68 (MHV68) as a mouse model for human gammaherpesviruses, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Our studies indicate that herpesvirus vUNG plays a critical role in pathogenesis, as vUNG deficient virus has major replication and infectivity defects. Gammaherpesvirus infects germinal center (GC) B cells and establishes latency in memory B cells, a process that can lead to immortalization and transformation of B cells. In GC B cells, host UNG activity process AID induced uracils to trigger antibody diversification, a process that also triggers chromosome translocations that are associated with lymphomas. Our studies indicate vUNG can process uracil in genomic DNA but with repair outcome different than host UNG. vUNG suppresses antibody mutation and class switch recombination in a manner that is both catalytic and non-catalytic dependent suggesting a scaffold function for vUNG. Thus, vUNG has the potential to contribute to altered viral pathogenesis, immunity and malignancy. We propose studies to understand how vUNG promotes viral pathogenesis and subverts host antibody diversification. In Aim 1, the structural and biochemical basis for error-free repair of DNA lesions by vUNG will be investigated. Differences between vUNG and host UNG will be characterized to determine the domains and interacting partners that result in differential error-free versus error-prone repair outcome. In Aim 2, the mechanisms by which vUNG promotes gammaherpesvirus pathogenesis will be investigated. The contribution of vUNG enzymatic function and scaffold functions will be characterized. In Aim 3, the impact of gammaherpesvirus infection and vUNG on B cell development will be investigated. B cell activation and immunoglobulin repertoire will be examined during MHV68 infection. Antibody repertoire and self-reactivity will be analyzed during acute infection and latency reactivation. Our aims straddle molecular immunology and molecular virology in the fields of gammaherpesvirus pathogenesis and B cell biology. The proposed studies should elucidate the mechanism by which vUNG supports viral replication, infection, and viral subversion of immunoglobulin diversification.