Mechanisms of hepatitis B virus cccDNA formation

项目概要 慢性乙型肝炎病毒 (HBV) 感染每年导致 887,000 人死亡。治愈的主要挑战 HBV 是指病毒基因组中稳定的共价闭合环状 DNA (cccDNA) 形式的根除， 其产生取决于难以捉摸的宿主因素。使用酵母提取物筛选，我们确定了五个核心 滞后链合成的成分 – PCNA、复制因子 C (RFC) 复合物、DNA 聚合酶 δ (POLδ)、FEN-1 和 DNA 连接酶 1 (LIG1) – 对于 cccDNA 形成至关重要。我们重建了cccDNA 与纯化的人类同系物形成，将它们建立为必要的最小因素集，并且 足以形成 cccDNA。我们进一步证明，抑制 POLδ 会显着降低 cccDNA 形成。在本提案中，我们将根据这些发现来确定精确的动力学 cccDNA 的形成，描绘了每个因素在修复过程每个步骤中的作用。在了解 rcDNA 到 cccDNA 修复的动力学，我们可以识别可能新颖的潜在限速步骤 破坏 cccDNA 形成和维持的治疗靶点。采用一系列创新技术 在细胞培养和小鼠模型系统中，我们将能够在生理相关方面测试我们的发现 将增强我们数据影响力的平台。发现对 rc-to cccDNA 至关重要的因素 这些系统中的转化将通过降解决定子介导的方法被破坏，该方法将允许微调 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的影响 打开慢性感染细胞中 cccDNA 的形成或已建立的 cccDNA 池的维持。至 为了提高此类研究的分辨率，我们还将在单细胞水平上检查表达水平如何 给定因子的值与 cccDNA 的值相关。总而言之，这些数据将为我们提供更全面的信息 这一过程对于慢性感染者体内乙型肝炎病毒的持续存在至关重要。

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.