Structural characterization of native HBV capsids and virions from human cells

人类细胞天然 HBV 衣壳和病毒颗粒的结构表征

• 批准号: 10736669
• 负责人: Che-Yen Wang
• 金额: $ 72.8万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-14 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736669
• 关键词: Address; Animal Experiments; Antiviral Agents; Arginine; Biology; C-terminal; Capsid; Capsid Proteins; Cell Culture System; Cell Culture Techniques; Cells; Classification; Complex; Cryoelectron Microscopy; Cytosol; Data; Disease; Double Stranded DNA Virus; Electron Microscope; Embryo; Ensure; Environment; Enzymes; Escherichia coli; Exhibits; Future; Genome; Hepatitis B Therapy; Hepatitis B Virus; Human; Hydrophobicity; In Vitro; Infection; Kidney; Knowledge; Lipids; Location; Mammalian Cell; Medical; Membrane Proteins; Methodology; Modeling; Molecular; Molecular Conformation; Molecular Structure; Molecular Virology; Mutation; N-terminal; Nucleic Acid Binding; Nucleic Acids; Pathogenesis; Phosphorylation; Physiological; Polymerase; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Proteins; Public Health; Publishing; RNA; RNA-Directed DNA Polymerase; Reaction; Resolution; Reverse Transcription; Signal Transduction; Single-Stranded DNA; Structure; System; Vaccines; Viral; Viral Envelope Proteins; Viral Genome; Viral Reverse Transcription; Virion; Virus Diseases; Virus Replication; antiviral drug development; design; ds-DNA; effective therapy; hepatoma cell; insight; mutant; novel; novel therapeutics; particle; premature; prevent; protein structure; reconstitution; response; structural determinants; trafficking; Address; Animal Experiments; Antiviral Agents; Arginine; Biology; C-terminal; Capsid; Capsid Proteins; Cell Culture System; Cell Culture Techniques; Cells; Classification; Complex; Cryoelectron Microscopy; Cytosol; Data; Disease; Double Stranded DNA Virus; Electron Microscope; Embryo; Ensure; Environment; Enzymes; Escherichia coli; Exhibits; Future; Genome; Hepatitis B Therapy; Hepatitis B Virus; Human; Hydrophobicity; In Vitro; Infection; Kidney; Knowledge; Lipids; Location; Mammalian Cell; Medical; Membrane Proteins; Methodology; Modeling; Molecular; Molecular Conformation; Molecular Structure; Molecular Virology; Mutation; N-terminal; Nucleic Acid Binding; Nucleic Acids; Pathogenesis; Phosphorylation; Physiological; Polymerase; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Proteins; Public Health; Publishing; RNA; RNA-Directed DNA Polymerase; Reaction; Resolution; Reverse Transcription; Signal Transduction; Single-Stranded DNA; Structure; System; Vaccines; Viral; Viral Envelope Proteins; Viral Genome; Viral Reverse Transcription; Virion; Virus Diseases; Virus Replication; antiviral drug development; design; ds-DNA; effective therapy; hepatoma cell; insight; mutant; novel; novel therapeutics; particle; premature; prevent; protein structure; reconstitution; response; structural determinants; trafficking

PROJECT SUMMARY Hepatitis B virus (HBV) infection is a global public health concern. Despite effective vaccines to prevent this disease, current approved treatment rarely leads to a complete cure. There is an unmet medical need for developing new therapeutics for HBV infection that can lead to a sustained response. Targeting HBV capsid assembly process has become an emerging strategy for developing new antiviral treatment for HBV. However, many efforts have been made by using different capsid protein (Cp) constructs expressed in Escherichia coli to mimic or reconstitute native-like viral particles. Yet, these structures cannot correctly represent the native HBV conformations due to the lacks of nucleic acid binding domain of the Cp, viral genome, and viral enzymes, all of which are required for viral replication. Furthermore, the lack of post translational modifications of the Cp also hampers the interpretation between observed Cp structures to the biomedical data obtained from the mammalian cell culture system or experimental animals. To date there is no available high-resolution native HBV structures, which is a major gap in knowledge of the HBV field. In this proposal, we aim to use cryo-electron microscope (cryo-EM) to directly characterize the structures of native HBV capsids and virions from human cells. In Aim 1, we will determine the high-resolution structures of purified intracellular HBV capsids with different types of viral genome. We will address the key questions concerning the structural dynamics of HBV capsids during genome maturation. We will also determine the structure of the HBV reverse transcriptase (RT) and its location during reverse transcription to help understand its mode of action (whether the RT is static or moves). In Aim 2, we will investigate the high-resolution structures of secreted HBV virions. This aim will address the questions concerning how HBV capsids interact with the viral envelop proteins. Finally, experimental findings from these two Aims will be integrated to elucidate capsid dynamics during HBV replication and illuminate the molecular determinant(s) of HBV envelopment. This proposal is expected to solve 4 types of intracellular HBV capsid structures (empty, RNA-filled, single- stranded DNA-filled, and mature partially double-stranded DNA-filled capsids) and 3 secreted enveloped HBV virion structures (empty, mature, and prematurely secreted virions) using cryo-EM to define the conformational changes of the capsid during viral replication, particularly in the context of different viral genome forms and interactions between the capsid and surface proteins. The methodology exploits appropriate mutations of Cp and RT to ensure obtaining homogenous particles of the various types as described above, which can be further computationally classified to minimize cross-contamination. Understanding the native HBV structures will provide valuable new information for HBV biology and guide the design of novel antiviral drugs in the future. The project is anticipated to impact fields ranging from HBV, molecular virology, antiviral drug development, and macromolecular structure and function.

项目摘要 丙型肝炎病毒（HBV）感染是全球公共卫生问题。尽管有效的疫苗可以防止这种情况 疾病，当前批准的治疗很少导致完全治愈。有未满足的医疗需求 为HBV感染开发新的治疗剂，从而导致持续反应。靶向HBV CAPSID 组装过程已成为为HBV开发新的抗病毒治疗的新兴策略。然而， 通过使用在大肠杆菌中表达的不同衣壳蛋白（CP）构建体进行的许多努力 模仿或重建天然的病毒颗粒。但是，这些结构无法正确表示本地HBV 由于缺乏CP，病毒基因组和病毒酶的核酸结合结构域而引起的构象，所有这些 病毒复制所需的。此外，缺乏CP的翻译后修改 阻碍观察到的CP结构对从哺乳动物获得的生物医学数据之间的解释 细胞培养系统或实验动物。迄今为止，没有可用的高分辨率本机HBV结构， 这是HBV领域知识的主要差距。 在此提案中，我们旨在使用冷冻电子显微镜（Cryo-EM）直接表征 来自人类细胞的天然HBV衣壳和病毒体。在AIM 1中，我们将确定 具有不同类型的病毒基因组的纯化细胞内HBV衣壳。我们将解决关键问题 关于基因组成熟过程中HBV衣壳的结构动力学。我们还将确定 HBV逆转录酶（RT）及其在逆转录过程中的位置的结构，以帮助理解 它的作用方式（RT是静态的还是静态的）。在AIM 2中，我们将研究高分辨率结构 分泌的HBV病毒体。此目标将解决有关HBV Capsids如何与病毒相互作用的问题 包络蛋白质。最后，这两个目标的实验发现将集成以阐明capsid HBV复制过程中的动力学并照亮了HBV包膜的分子决定因素。 预计该建议将解决4种类型的细胞内HBV衣壳结构（空，RNA充满RNA，单个 滞留的DNA填充和成熟的部分双链DNA填充的衣壳）和3个分泌的包裹HBV 使用冷冻EM定义构象的病毒体结构（空，成熟和过早分泌的病毒体） 病毒复制过程中衣壳的变化，特别是在不同的病毒基因组形式的背景下 衣壳和表面蛋白之间的相互作用。该方法利用了CP的适当突变 和RT，以确保获得上述各种类型的同质颗粒，可以进一步 在计算上分类以最大程度地减少交叉污染。 了解本地HBV结构将为HBV生物学提供有价值的新信息，并指导 未来新型抗病毒药物的设计。预计该项目将影响HBV的领域 分子病毒学，抗病毒药物发育以及大分子结构和功能。