Molecular Mechanism of Hepadnavirus Persistence

嗜肝DNA病毒持久性的分子机制

• 批准号: 7569354
• 负责人: Jianming Hu
• 金额: $ 30.3万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7569354
• 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; Chronic Hepatitis B; Chronic viral hepatitis; Circular DNA; Cirrhosis; Conversion disorder; Coupled; DNA; DNA Repair; DNA repair protein; Defect; Development; Duck Hepatitis B Virus; Ducks; 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; 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; Simian B disease; Staging; Structure; Testing; Viral; Viral Genes; Viral Reverse Transcription; Virion; abstracting; base; combinatorial; ds-DNA; homologous recombination; human disease; insight; multicatalytic endopeptidase complex; novel; pgRNA; reconstitution; repaired; terminal redundancy; viral DNA

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.

描述（由申请人提供）：B肝炎病毒（HBV）仍然是慢性肝病（包括肝硬化和癌症）的全球病因。目前慢性B型肝炎的抗病毒治疗仅部分有效。特别是，附加型病毒DNA，即所谓的共价闭合环状（CCC）DNA，即使在多年的抗病毒治疗后仍存在于感染的细胞核中。CCC DNA作为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是治愈HBV感染的先决条件。CCC DNA由病毒基因组DNA产生，其具有松弛环状（RC）、部分双链结构。为了完成RC到CCC的DNA转化过程，必须发生多个生化反应，目前对此还不清楚。本申请的总体目标是开始分析CCC DNA形成的分子机制，使用HBV和鸭HBV（DHBV）作为模型系统。提出了三个具体目标。具体目标1将是确定潜在的途径，包括推定的中间体，CCC DNA的形成。使用体外细胞培养系统，HBV和DHBV CCC DNA的形成发生和潜在的中间体积累，我们计划确定和详细描述这些中间体。这一点，再加上具体目标2和3中提出的干扰其生产的定向方法，将为CCC DNA形成的潜在途径提供重要线索。具体目标2将确定特定病毒因子的作用，即，病毒包膜和逆转录酶蛋白，在CCC DNA的形成和调节，采用遗传和生物化学方法的组合。具体目标3将确定选定的宿主因子，特别是细胞DNA修复因子，在CCC DNA形成的作用，使用现有的细胞培养系统和无细胞检测，将开发。这些研究将为CCC DNA形成机制带来急需的见解，这可能有助于开发直接针对病毒复制这一关键步骤的新型抗病毒药物。此外，它们可能为细胞DNA损伤修复机制提供新的线索，该机制的故障是从发育缺陷到癌症的各种严重人类疾病的基础。公共卫生相关性：B型肝炎病毒（HBV）是慢性肝病（包括肝硬化和肝癌）的全球性病因。我们建议阐明的机制，病毒和宿主因素参与，产生核附加型病毒DNA，这是HBV持久性的分子基础。这些研究将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性感染的新型抗病毒药物。