Uracil DNA Glycosylases in Gammaherpesvirus Pathogenesis and B Cell Development

项目摘要 每种疱疹病毒编码一种哺乳动物尿嘧啶DNA糖基酶(Vung)的病毒同源物。哺乳动物UNG是 一种酶，能去除错误结合和致突变的尿嘧啶，留下一个基本的部位，通常由 宿主碱基切除修复途径。尽管存在保守的序列和体外活性 Vung和宿主UNG，对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复制的组成部分相互作用 机制；然而，它在原代细胞后期复制事件中的作用尚不确定。我们利用小鼠 伽马疱疹病毒68(MHV68)作为人类伽马疱疹病毒、Epstein-Barr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 发病机制，由于Vung缺陷病毒具有主要的复制和传染性缺陷。伽马疱疹病毒感染 生发中心(GC)B细胞，并在记忆B细胞中建立潜伏期，这一过程可能导致 B细胞的永生化和转化。在GC B细胞中，宿主UNG活动过程AID诱导尿嘧啶 引发抗体多样化，这一过程也会触发与以下相关的染色体易位 淋巴瘤。我们的研究表明，Vung可以在基因组DNA中处理尿嘧啶，但修复结果不同 而不是主办UNG。Vung以一种既能抑制抗体突变又能抑制类切换重组的方式 催化和非催化依赖，提示Vung具有支架功能。因此，冯有潜力 有助于改变病毒的发病机制、免疫力和恶性程度。我们建议进行研究，以了解 Vung促进病毒致病，颠覆宿主抗体多样化。在目标1中，结构和 Vung无差错修复DNA损伤的生化基础将被调查。两者之间的差异 Vung和东道主UNG将确定导致以下结果的域和互动伙伴 无错误修复结果与易出错修复结果的差异。在目标2中，冯推动 将对伽马疱疹病毒的发病机制进行调查。Vung酶功能的贡献和 将对脚手架的功能进行表征。在目标3中，伽马疱疹病毒感染和Vung对B 将对细胞发育进行调查。B细胞活化和免疫球蛋白谱系将在 MHV68感染。将分析急性感染和潜伏期的抗体谱系和自身反应性 重新激活。我们的目标是跨越分子免疫学和分子病毒学领域 伽马疱疹病毒的发病机制和B细胞生物学。拟议的研究应通过以下方式阐明这一机制 其中Vung支持病毒复制、感染和病毒颠覆免疫球蛋白多样化。

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.