The Structural Biology of HBV

乙型肝炎病毒的结构生物学

• 批准号: 9899197
• 负责人: Adam Zlotnick
• 金额: $ 37.3万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899197
• 关键词: Address; Adopted; Antiviral Agents; Basic Science; Binding; Binding Sites; Biochemistry; Biophysics; Capsid; Cell Culture Techniques; Cell Nucleus; Cell secretion; Chronic Hepatitis B; Complex; Core Assembly; Core Protein; Coupled; Cryoelectron Microscopy; DNA; DNA-Directed RNA Polymerase; Data; Equipment; Estrogen receptor positive; Genome; Goals; Gold; HepG2; Hepadnaviridae; Hepatitis B; Hepatitis B Virus; Importins; In Vitro; Infection; Ligands; Liver; Location; Membrane; Membrane Proteins; Molecular Conformation; Noise; Nuclear Import; Nucleic Acids; Participant; Pattern; Peptide Hydrolases; Peptides; Polymerase; Polymers; Process; Property; Proteins; RNA; RNA Binding; Reaction; Reporting; Resolution; Reverse Transcriptase Inhibitors; Reverse Transcription; Signal Transduction; Site; Stimulus; Structural Biochemistry; Structure; Surface; Surface Antigens; System; Techniques; Technology; Testing; Time; Travel; Viral; Viral Genome; Viral Load result; Virion; Virus; Virus Diseases; biophysical techniques; chronic infection; density; ds-DNA; env Gene Products; image reconstruction; improved; in vivo; nucleic acid structure; particle; reconstruction; single molecule; standard of care; stem; structural biology; viral RNA

Summary 240 million people suffer from chronic Hepatitis B Virus infection (HBV). In vivo, assembly of new virions begins with a complex of the viral polymerase with a stem loop on the viral RNA. This complex nucleates assembly of a T=4 capsid resulting in the RNA-filled core. Within the RNA-filled core the polymerase becomes active and reverse transcribes the linear single stranded RNA pregenome to the relaxed circular double strand DNA of mature HBV cores. Mature cores and empty cores, but not immature cores, display signals that allow their transport to the nucleus or the ER. These directions respectively maintain chronic infection or result in envelopment and secretion from the cell. Our preliminary studies show that viral RNA lines the interior surface of the capsid and supports the hypothesis that in RNA-filled cores the nucleating polymerase complex occupies a specific site (or a small number of sites) with respect to the capsid. We further propose that that the polymerase travels on the RNA during reverse transcription. We propose that owing to its stiffness, the dsDNA genome of mature capsids must adopt a completely different organization. We will investigate the structure and biochemistry of empty, RNA-filled, and mature DNA-filled capsid. Our overarching goals are to describe the mechanism of reverse transcription and the structural basis for signaling maturation of the HBV genome. Towards this end we will develop purification strategies for purifying different classes of core, examine their dynamic properties using biophysical techniques, use single molecule techniques to determine variability of core mass, and investigate the reverse transcription reaction itself. In a second set of aims we will determine structures of RNA-filled and DNA-filled cores to investigate the disposition of the nucleic acid, its effects on capsid symmetry, and the availability of capsid binding sites for host proteins, such as the importins responsible for nuclear import. Treatment of chronic HBV with reverse transcriptase inhibitors, the standard of care, decreases viral load and improves liver condition but it rarely leads to a “cure”, even after years of treatment. Remarkably, there is no real understanding of the structural basis of reverse transcription and how a signal for nucleic acid state is transduced to allow the virus lifecycle to progress. Addressing these deficits is the goal of this proposal.

概括 2.4 亿人患有慢性乙型肝炎病毒 (HBV) 感染。在体内，组装新的 病毒粒子起始于病毒聚合酶与病毒RNA上的茎环的复合体。这个综合体 使 T=4 衣壳组装成核，形成充满 RNA 的核心。在充满 RNA 的核心内 聚合酶变得活跃并将线性单链RNA前基因组逆转录为 成熟 HBV 核心的松弛环状双链 DNA。成熟核和空核，但不是 未成熟的核心会显示信号，允许它们转运到细胞核或内质网。这些方向 分别维持慢性感染或导致细胞包封和分泌。我们的 初步研究表明，病毒 RNA 排列在衣壳的内表面并支持 假设在充满 RNA 的核心中，成核聚合酶复合物占据特定位点（或 少量位点）相对于衣壳。我们进一步建议聚合酶传播 逆转录过程中RNA的作用。我们认为，由于其刚性，dsDNA 基因组 成熟的衣壳必须采用完全不同的组织。我们将研究结构和 空的、RNA 填充的和成熟 DNA 填充的衣壳的生物化学。我们的总体目标是 描述逆转录机制和信号成熟的结构基础 乙肝病毒基因组。为此，我们将制定纯化策略来纯化不同的物质 核心类别，使用生物物理技术检查其动态特性，使用单分子 确定核心质量变异性并研究逆转录反应的技术 本身。在第二组目标中，我们将确定 RNA 填充和 DNA 填充核心的结构，以 研究核酸的处置、其对衣壳对称性的影响以及 宿主蛋白的衣壳结合位点，例如负责核输入的输入蛋白。 使用逆转录酶抑制剂治疗慢性乙型肝炎（护理标准）可减少病毒感染 负荷并改善肝脏状况，但即使经过多年的治疗，它也很少导致“治愈”。 值得注意的是，人们对逆转录的结构基础以及逆转录如何发生并没有真正的了解。 核酸状态的信号被转导以允许病毒生命周期继续进行。解决这些问题 赤字是本提案的目标。