Mechanisms of hepatitis B virus cccDNA formation

项目总结 慢性乙肝病毒感染每年导致88.7万人死亡。治愈的主要挑战 乙肝病毒是对稳定的共价闭合环状DNA(CcDNA)形式的病毒基因组的根除，它 它的产生依赖于难以捉摸的宿主因素。使用酵母抽提物筛选，我们鉴定了五个核心 滞后链合成的组成部分--增殖细胞核抗原、复制因子C复合体、脱氧核糖核酸聚合酶δ (POLδ)、FEN-1和DNA连接酶1(LIG1)-作为cccDNA形成所必需的。我们重组了cccDNA 与纯化的人类同源物形成，建立这些作为必要的最小因素集和 足以形成cccDNA。我们进一步证明，抑制POLδ显著降低 CccDNA形成。在这项提案中，我们将以这些发现为基础来确定 CccDNA的形成，描绘了每个因子在修复过程的每一步中的作用。在理解 的动态，我们可以确定潜在的限速步骤，可能是新的 干扰cccDNA形成和维持的治疗靶点。使用一系列创新技术 在细胞培养和小鼠模型系统中，我们将能够测试我们的发现在生理上的相关性 这些平台将加强我们数据的影响。发现RC-to-cccDNA的关键因素 这些系统中的转换将被允许微调的退化介导性方法破坏 控制表达以减轻任何潜在的细胞毒性。然后，我们可以监控每个因素对 在慢性感染细胞中打开cccDNA形成或维护已建立的cccDNA池。至 提高这类研究的分辨率，我们还将在单细胞水平上考察表达水平如何 与cccDNA相关的某一特定因子。总而言之，这些数据将为我们提供更全面的 认为这一过程对慢性感染者体内乙肝病毒的持久性至关重要。

Project summary Chronic hepatitis B virus (HBV) infection results in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which depends on elusive host factors for its generation. Using a yeast extract screen, we identified five core components of lagging strand synthesis –PCNA, the replication factor C (RFC) complex, DNA polymerase δ (POLδ), FEN-1, and DNA ligase 1 (LIG1) – as essential for cccDNA formation. We reconstituted cccDNA formation with purified human homologs, establishing these as a minimal set of factors necessary and sufficient for cccDNA formation. We further demonstrated that inhibiting POLδ significantly diminishes cccDNA formation. In this proposal, we will build on these findings to determine the precise kinetics of cccDNA formation, delineating the role of each factor at every step of the repair process. In understanding the dynamics of rcDNA to cccDNA repair, we can identify potential rate-limiting steps that could be novel therapeutic targets for disrupting cccDNA formation and maintenance. Using a series of innovative techniques in both cell culture and mouse model systems, we will be able to test our findings in physiologically relevant platforms that will strengthen the impact of our data. Factors found to be critical for rc- to cccDNA conversion will be disrupted in these systems by a degron-mediated approach that will allow for fine-tuned control of expression to alleviate any potential cytotoxicity. We can then monitor the effect of each factor in turn on cccDNA formation or the maintenance of established cccDNA pools in chronically infected cells. To increase the resolution of such studies, we will also examine at the single-cell level how the expression levels of a given factor correlate with that of cccDNA. Altogether, these data will give us a far more comprehensive view of this process critical to the persistence of HBV in chronically infected individuals.