Molecular Mechanisms of HBV cccDNA Formation

HBV cccDNA 形成的分子机制

• 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
• 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

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.

摘要 B型肝炎病毒（HBV）共价闭合环状（ccc）DNA在病毒感染的建立中起着重要作用。 感染和持久性，并且是停止治疗后病毒反弹的基础，以及 即使用目前批准的药物进行延长治疗，也难以治愈。HBV cccDNA是 通过部分双链松弛环状（rc）DNA的转化在初始感染时建立 通过利用宿主细胞的DNA修复机制， 在细胞核中。cccDNA附加体水平通过复制周期维持，所述复制周期涉及 将cccDNA转录物（称为前基因组RNA）逆转录到子代rcDNA基因组中， 其返回细胞核以转化成cccDNA。将rcDNA转化为cccDNA需要 从DNA链之一的5'末端去除聚合酶的共价连接拷贝，并且这 “脱蛋白”步骤产生DNA中间体，脱蛋白的rcDNA（DP-rcDNA），其作为前体， cccDNA形成。rcDNA脱蛋白是HBV核衣壳转运的触发信号 将含有成熟病毒DNA的RNA插入细胞核，在细胞核中发生rcDNA到cccDNA的转化。我们有 最近绘制了胞质DP-rcDNA的末端，这表明病毒聚合酶和RNA 在脱蛋白过程中，引物完全从rcDNA中除去，正链DNA进一步延长， DP-rcDNA上保留了末端冗余序列。此外，我们和其他人最近的研究表明， 已经鉴定了一些参与cccDNA形成的宿主DNA修复因子。有很多 为了更好地理解cccDNA生物合成，还需要阐明分子细节， HBV感染的免疫活性小动物模型的建立受到以下因素的阻碍： 小鼠肝细胞中cccDNA的形成。在这项研究应用中，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 DP-rcDNA（Aim 1）和cccDNA（Aim 2）的生物发生机制，并定义宿主 小鼠肝细胞中cccDNA形成失败的决定因素（目的3）。我们的最终目标是 阐明了HBV cccDNA形成的分子机制/途径的连贯画面。的 该项目的完成将揭示治疗B型肝炎的新的潜在抗病毒靶点， 开发对HBV感染完全易感的小鼠模型。