Molecular Mechanisms of HBV cccDNA Formation

HBV cccDNA 形成的分子机制

基本信息

批准号：
10313040
负责人：
Haitao Guo
金额：
$ 38.25万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-11 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10313040
关键词：
Adverse effects Affect Anabolism Antiviral Agents Binding Biochemistry Biogenesis Biology Cell Line Cell Nucleus Cell physiology Cells Chemicals Chronic Chronic Hepatitis B Circular DNA Cirrhosis Complement Complex Cytoplasm DNA DNA Ligases DNA Repair DNA Repair Gene Data Development Drug resistance Employment Episome Excision Exhibits Failure Flap Endonucleases Genes Genetic Transcription Genome Genomics Goals Guanosine HBV Animal Model Hepatitis B Therapy Hepatitis B Virus Hepatocyte Human Immunocompetent Individual Infection Interferon-alpha Life Cycle Stages Link Liver Longevity Maintenance Malignant neoplasm of liver Maps Mass Spectrum Analysis Messenger RNA Minor Molecular Molecular Biology Mus Mutagenesis Nature Nuclear Nucleocapsid Nucleotide Excision Repair Pathway interactions Pharmaceutical Preparations Pharmacotherapy Play Polymerase Primary carcinoma of the liver cells Process Production Proteins Proteome Proteomics Public Health RNA RNA primers Reporting Research Reverse Transcription Risk Factors Role Side Signal Transduction Study models Technology TimeLine Transcript Transportation Viral Viral Genome Virus Virus Diseases Virus Inhibitors Virus Replication Work analog chromatin immunoprecipitation comparative effective therapy high risk insight knock-down mouse model novel novel therapeutics phosphodiester phosphoric diester hydrolase receptor response stable cell line targeted treatment terminal redundancy transcriptome tyrosyl-DNA phosphodiesterase viral DNA viral genomics viral rebound

项目摘要

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (ccc) DNA plays a central role in the establishment of viral infection and persistence, and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment with current approved medications. HBV cccDNA is established upon initial infection through conversion of the partially double stranded relaxed circular (rc) DNA viral genome containing terminal peculiarities, through employment of the host cell’s DNA repair mechanisms in the nucleus. The cccDNA episome levels are maintained through a replication cycle that involves retrotranscription of a cccDNA transcript, termed pregenomic RNA, into progeny rcDNA genomes, some of which are returned to the nucleus for conversion into cccDNA. The conversion of rcDNA into cccDNA requires the removal of a covalently-linked copy of the polymerase from the 5’ end of one of the DNA strands, and this “deproteination” step generates a DNA intermediate, the deproteinated rcDNA (DP-rcDNA), as precursor for cccDNA formation. The rcDNA deproteination is a trigger signal for transportation of HBV nucleocapsid containing mature viral DNA into nucleus, where the rcDNA to cccDNA conversion takes place. We have recently mapped the termini of cytoplasmic DP-rcDNA, which demonstrated that the viral polymerase and RNA primer are completely removed from rcDNA during deproteination, the plus strand DNA is further elongated but the terminal redundant sequence is maintained on DP-rcDNA. In addition, recent studies by us and others have identified a handful of host DNA repair factors involved in cccDNA formation. However, there are many molecular details yet to be elucidated for a better understanding of cccDNA biosynthesis, and the establishment of immunocompetent small animal model for HBV infection is hampered by the inability of cccDNA formation in mouse hepatocyte. In this research application, by making use of a battery of molecular biology, biochemistry, proteomics and genomics technologies, we propose to further elucidate the molecular mechanisms underlying the biogenesis of DP-rcDNA (Aim 1) and cccDNA (Aim 2), and to define the host determinant(s) for the failure of cccDNA formation in mouse hepatocyte (Aim 3). Our ultimate goal is to illustrate a coherent picture of the molecular mechanisms/pathway for HBV cccDNA formation. The accomplishment of this project will reveal new potential antiviral targets for treatment of hepatitis B and aid the development of a mouse model fully susceptible to HBV infection.

抽象的乙型肝炎病毒 (HBV) 共价闭合环状 (ccc) DNA 在病毒的建立中发挥着核心作用感染和持续存在，是停止治疗后病毒反弹的基础，也是即使在使用当前批准的药物进行长期治疗后，治愈方法仍然难以实现。通过部分双链松弛环状 (rc) DNA 的转化在初始感染后建立通过利用宿主细胞的 DNA 修复机制，病毒基因组包含末端特性 cccDNA 附加体水平通过涉及的复制周期得以维持。 cccDNA 转录物（称为前基因组 RNA）逆转录成后代 rcDNA 基因组，其中一些返回细胞核转化为 cccDNA 将 rcDNA 转化为 cccDNA 需要。从 DNA 链之一的 5' 端去除共价连接的聚合酶副本，并且这 “去蛋白”步骤生成 DNA 中间体，即去蛋白 rcDNA (DP-rcDNA)，作为 cccDNA 的形成是 HBV 核衣壳运输的触发信号。含有成熟病毒 DNA 进入细胞核，在那里发生 rcDNA 到 cccDNA 的转换。最近绘制了细胞质 DP-rcDNA 的末端图谱，这表明病毒聚合酶和 RNA 在去蛋白过程中，引物从 rcDNA 中完全去除，正链 DNA 进一步拉长，但此外，我们和其他人最近的研究还保留了末端冗余序列。已经鉴定出少数参与 cccDNA 形成的宿主 DNA 修复因子。为了更好地理解 cccDNA 生物合成，分子细节尚待阐明，并且乙型肝炎病毒感染的免疫活性小动物模型的建立因缺乏免疫能力而受到阻碍小鼠肝细胞中 cccDNA 的形成在本研究应用中，通过使用一组分子。生物学、生物化学、蛋白质组学和基因组学技术，我们建议进一步阐明分子生物学 DP-rcDNA（目标 1）和 cccDNA（目标 2）生物发生的机制，并定义宿主小鼠肝细胞中 cccDNA 形成失败的决定因素（目标 3）。阐明了 HBV cccDNA 形成的分子机制/途径的连贯图景。该项目的完成将揭示治疗乙型肝炎的新的潜在抗病毒靶标，并帮助开发完全易受乙型肝炎病毒感染的小鼠模型。