The Structural Biology of HBV

乙型肝炎病毒的结构生物学

• 批准号: 10372082
• 负责人: Adam Zlotnick
• 金额: $ 37.84万
• 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 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372082
• 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; Genome; Goals; Gold; HepG2; Hepadnaviridae; Hepatitis B Infection; 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; Persons; Polymerase; Polymers; Process; Property; Proteins; RNA; RNA Binding; Reaction; Reporting; Resolution; Reverse Transcriptase Inhibitors; Reverse Transcription; Signal Transduction; Site; Stimulus; 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 2.4亿人患有慢性丙型肝炎病毒感染（HBV）。在体内，新组装 病毒体从病毒聚合酶的复合物开始，并在病毒RNA上带有茎环。这个复合物 成核会导致t = 4 capsid的组装产生RNA填充的核心。在充满RNA的核心内 聚合酶变得活跃，并将线性单链RNA前植物抄录到 成熟HBV核心的松弛圆形双链DNA。成熟的核心和空的核心，但没有 未成熟的核心，显示信号，允许它们运输到核或ER。这些方向 分别维持慢性感染或导致细胞的环境和分泌。我们的 初步研究表明，病毒RNA在衣壳的内部表面线条并支持 假设在富含RNA的核中，成核聚合酶复合酶占据了特定位点（或A 少数站点）相对于衣壳。我们进一步提出，聚合酶会旅行 在逆转录过程中的RNA上。我们提出，由于其刚度，DSDNA的基因组 成熟的衣壳必须采用一个完全不同的组织。我们将研究结构和 空的，RNA充满RNA和成熟的DNA填充衣壳的生物化学。我们的总体目标是 描述逆转录的机制和信号成熟的结构基础 HBV基因组。为此，我们将制定净化策略来净化不同的 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充满RNA和DNA填充核的结构 研究核酸的处置，其对衣壳对称性的影响以及可用性 宿主蛋白的衣壳结合位点，例如负责核进口的进口素。 用逆转录酶抑制剂（护理标准）治疗慢性HBV的病毒 负载并改善肝脏状况，但即使经过多年的治疗，也很少会导致“治愈”。 值得注意的是，对逆转录的结构基础没有真正的理解 核酸状态的信号被翻译成允许病毒生命周期的发展。解决这些 缺陷是该提议的目标。

项目成果

HBV Core Protein Is in Flux between Cytoplasmic, Nuclear, and Nucleolar Compartments.

• DOI: 10.1128/mbio.03514-20
• 发表时间: 2021-02-09
• 期刊: mBio
• 影响因子: 6.4
• 作者: Nair S;Zlotnick A
• 通讯作者: Zlotnick A

Virus self-assembly proceeds through contact-rich energy minima.

• DOI: 10.1126/sciadv.abg0811
• 发表时间: 2021-11-05
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Buzón P;Maity S;Christodoulis P;Wiertsema MJ;Dunkelbarger S;Kim C;Wuite GJL;Zlotnick A;Roos WH
• 通讯作者: Roos WH