Molecular Mechanism of Hepadnavirus Persistence

嗜肝DNA病毒持久性的分子机制

• 批准号: 8240404
• 负责人: Jianming Hu
• 金额: $ 29.72万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240404
• 关键词: Affect; Antiviral Agents; Antiviral Therapy; Biochemical; Biochemical Pathway; Biochemical Reaction; Biological Assay; Biological Models; Cell Culture System; Cell Extracts; Cell Nucleus; Cell-Free System; Cells; Chronic Hepatitis B; Chronic viral hepatitis; Circular DNA; Cirrhosis; Conversion disorder; Coupled; DNA; DNA Repair; DNA repair protein; Defect; Development; Duck Hepatitis B Virus; Excision; Family; Gene Expression; Genetic; Genetic Transcription; Genome; Genomics; Goals; Hepadnaviridae; Hepatitis B Virus; Hepatocyte; In Vitro; Infection; Integration Host Factors; Ligation; Light; Link; Liver Cirrhosis; Liver diseases; Malignant Neoplasms; Malignant neoplasm of liver; Membrane Proteins; Molecular; Mutagenesis; Nuclear; Nucleocapsid; Pathway interactions; Primary carcinoma of the liver cells; Process; Production; Protein Analysis; Proteins; RNA; RNA Caps; RNA-Directed DNA Polymerase; Regulation; Relax transcriptional regulator; Research; Resistance; Retroviridae; Reverse Transcription; Risk; Role; Staging; Structure; Testing; Viral; Viral Genes; Viral Reverse Transcription; Virion; Virus Diseases; abstracting; base; chronic liver disease; combinatorial; ds-DNA; homologous recombination; human disease; insight; multicatalytic endopeptidase complex; novel; pgRNA; reconstitution; repaired; terminal redundancy; viral DNA

Abstract The hepatitis B virus (HBV) remains a global cause of chronic liver diseases, including liver cirrhosis and cancer. Current antiviral therapy for chronic hepatitis B is only partially effective. In particular, the episomal viral DNA, the so-called covalently closed circular (CCC) DNA, persists in the infected cell nucleus even after years of antiviral treatment. The CCC DNA serves as the template for all viral transcriptions and is the molecular basis of HBV persistence. Therefore, the elimination of the CCC DNA is a prerequisite for any curing of an HBV infection. The CCC DNA is generated from the viral genomic DNA, which has a relaxed circular (RC), partially double-stranded structure. To complete the RC to CCC DNA conversion process, multiple biochemical reactions have to occur, about which nothing is currently understood. The overall goal of the current application is to begin to analyze the molecular mechanisms of CCC DNA formation, using both HBV and the duck HBV (DHBV) as model systems. Three Specific Aims are proposed. Specific Aim 1 will be to determine the potential pathways, including putative intermediates, of CCC DNA formation. Using in vitro cell culture systems where HBV and DHBV CCC DNA formation takes place and potential intermediates accumulate, we plan to identify and characterize these intermediates in detail. This, coupled with directed approaches to perturb their production as proposed in Specific Aims 2 & 3, will provide important clues about the potential pathways of CCC DNA formation. Specific Aim 2 will determine the role of specific viral factors, i.e., the viral envelope and reverse transcriptase proteins, in the formation and regulation of CCC DNA, employing a combination of genetic and biochemical approaches. Specific Aim 3 will determine the role of selected host factors, particularly cellular DNA repair factors, in CCC DNA formation, using both existing cell culture systems and cell-free assays that will be developed. These studies should bring much needed insights into the mechanism of CCC DNA formation, which may facilitate the development of novel antivirals targeted directly at this critical step of viral replication. In addition, they may shed new light on the mechanisms of cellular DNA damage repair, the malfunction of which underlies a variety of serious human diseases from developmental defects to cancer. Project Narrative The hepatitis B virus (HBV) is a global cause of chronic liver diseases, including liver cirrhosis and cancer. We propose to elucidate the mechanisms of, and viral and host factors involved in, producing the nuclear episomal viral DNA, which is the molecular basis of HBV persistence. These studies should facilitate the development of novel antiviral agents targeted directly at this critical step of viral replication and capable of curing persistent infections.

抽象的 乙型肝炎病毒 (HBV) 仍然是慢性肝病（包括肝硬化）的全球病因 和癌症。目前针对慢性乙型肝炎的抗病毒治疗仅部分有效。特别是，游离型 病毒 DNA，即所谓的共价闭合环状 (CCC) DNA，即使在感染后仍持续存在于受感染的细胞核中。 多年的抗病毒治疗。 CCC DNA 是所有病毒转录的模板，是 HBV 持续存在的分子基础。因此，消除 CCC DNA 是任何治愈的先决条件。 乙型肝炎病毒感染。 CCC DNA 由病毒基因组 DNA 产生，具有松弛的环状结构 (RC)，部分双链结构。为了完成 RC 到 CCC DNA 的转换过程，需要多个 生化反应必须发生，但目前对此还一无所知。该项目的总体目标是 目前的应用是开始分析CCC DNA形成的分子机制，使用HBV和 和鸭 HBV (DHBV) 作为模型系统。提出了三个具体目标。具体目标 1 是 确定 CCC DNA 形成的潜在途径，包括假定的中间体。使用体外细胞 HBV 和 DHBV CCC DNA 形成的培养系统以及潜在的中间体 积累后，我们计划详细识别和表征这些中间体。这一点，再加上定向 具体目标 2 和 3 中提出的扰乱其生产的方法将提供重要线索 CCC DNA 形成的潜在途径。具体目标 2 将确定特定病毒因素的作用， 即病毒包膜和逆转录酶蛋白在 CCC DNA 的形成和调节中， 采用遗传和生化方法的结合。具体目标 3 将确定以下角色： 使用现有细胞在 CCC DNA 形成中选择宿主因子，特别是细胞 DNA 修复因子 将开发的培养系统和无细胞测定。这些研究应该带来急需的见解 深入研究 CCC DNA 形成机制，可能有助于开发新型靶向抗病毒药物 直接作用于病毒复制的这一关键步骤。此外，它们可能对机制产生新的认识。 细胞 DNA 损伤修复，其故障是多种严重人类疾病的基础 发育缺陷导致癌症。项目叙述 乙型肝炎病毒 (HBV) 是导致慢性肝病的全球性原因，包括肝硬化和 癌症。我们建议阐明产生该病毒的机制以及参与的病毒和宿主因素 核游离病毒 DNA，这是 HBV 持续存在的分子基础。这些研究应该有助于 开发直接针对病毒复制这一关键步骤并能够 治愈持续感染。