Mechanisms of hepatitis B virus cccDNA formation

项目摘要 慢性B型肝炎病毒（HBV）感染每年导致887，000人死亡。治疗的核心挑战是 HBV是病毒基因组的稳定共价闭合环状DNA（cccDNA）形式的根除， 依赖于难以捉摸的宿主因子来产生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（LIG 1）-对于cccDNA形成是必需的。我们重组了cccDNA 与纯化的人类同源物形成，将这些确定为必要的最小因子集， 足以形成cccDNA。我们进一步证明，抑制POLδ可以显著降低 cccDNA形成。在本提案中，我们将基于这些发现来确定 cccDNA的形成，描绘了每个因子在修复过程的每个步骤中的作用。在理解 rcDNA到cccDNA修复的动力学，我们可以确定潜在的限速步骤， 用于破坏cccDNA形成和维持的治疗靶点。运用一系列创新技术 在细胞培养和小鼠模型系统中，我们将能够在生理学相关方面测试我们的发现。 这些平台将增强我们数据的影响力。发现对rc-至cccDNA至关重要的因素 在这些系统中，通过允许微调的degron介导的方法， 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， 启动cccDNA形成或维持慢性感染细胞中已建立的cccDNA库。到 为了提高这种研究的分辨率，我们还将在单细胞水平上研究表达水平如何影响细胞的生长。 与cccDNA相关。总之，这些数据将给我们一个更全面的 认为这一过程对慢性感染者体内HBV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.