Mechanisms of hepatitis B virus cccDNA formation

乙型肝炎病毒cccDNA形成机制

基本信息

批准号：
10165502
负责人：
Alexander Ploss
金额：
$ 55.41万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-15 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10165502
关键词：
Affect Animal Model Antibodies Antiviral Agents Antiviral Therapy Biochemical Biological Assay Biological Models Cell Culture Techniques Cell Extracts Cell Line Cell Nucleus Cells Cessation of life Chronic Chronic Hepatitis B Circular DNA Clustered Regularly Interspaced Short Palindromic Repeats Coculture Techniques Complex DNA Ligases DNA Repair DNA lesion DNA-Directed DNA Polymerase Data Exposure to Generations Genetic Genome Goals Health HepG2 Hepatitis B Virus Hepatocyte Human In Vitro Individual Infection Integration Host Factors Kinetics Knock-out Learning Lesion Life Cycle Stages Liver Liver diseases Maintenance Malignant neoplasm of liver Mediating Methods Molecular Monitor Mus Mutation Nature Pan Genus Patients Physiological Plant Roots Polymerase Process Public Health RTH-1 Nuclease Recombinants Refractory Regimen Resolution Risk Role Series Set protein System Techniques Testing Therapeutic Intervention Transcript Vaccination Viral Genome Virion Virus Virus Diseases Virus Receptors Work Yeasts cytotoxicity design experience hepatoma cell high risk innovation knock-down mouse model new therapeutic target novel overexpression pathogenic virus prevent purge reconstitution repair enzyme repaired replication factor C small hairpin RNA

项目摘要

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.

项目摘要慢性丙型肝炎病毒（HBV）感染每年导致887,000人死亡。治愈的核心挑战 HBV是病毒基因组的稳定闭合圆形DNA（CCCDNA）形式的稳定构造取决于其一代的难以捉摸的宿主因素。使用酵母提取物屏幕，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的形成至关重要。我们重组了CCCDNA 具有纯化的人类同源物的形成，将其确定为必要的最小因素集足以形成CCCDNA。我们进一步证明，抑制polδ会显着减少 CCCDNA形成。在此提案中，我们将基于这些发现，以确定 CCCDNA的形成，描绘了修复过程的每个步骤中每个因素的作用。在理解 rcDNA到CCCDNA修复的动力学，我们可以识别可能是新颖的潜在限制步骤破坏CCCDNA形成和维护的治疗靶标。使用一系列创新技术在细胞培养和鼠标模型系统中，我们将能够在物理相关的情况下测试我们的发现可以加强数据影响的平台。发现对RC-到CCCDNA至关重要的因素这些系统将通过Degron介导的方法中断转换，该方法将允许进行微调控制表达以减轻任何潜在的细胞毒性。然后，我们可以监视每个因素的影响打开CCCDNA形成或在慢性感染细胞中维持已建立的CCCDNA池。到增加了此类研究的分辨率，我们还将在单细胞水平上检查表达水平如何给定因子与CCCDNA的因子相关。总之，这些数据将使我们更加全面这一过程对于长期感染的个体中HBV持续存在至关重要的观点。