权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
10393606
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/10393606
关键词：
Affect Animal Model Antibodies 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 Persons 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.

项目总结