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

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 B型肝炎病毒（HBV）感染是一个全球性的公共卫生问题。尽管有有效的疫苗来预防这种情况， 然而，目前批准的治疗很少导致完全治愈。有一个未满足的医疗需求， 开发新的治疗HBV感染的药物，可以导致持续的反应。靶向HBV衣壳 组装过程已成为开发新的HBV抗病毒治疗的新兴策略。然而，在这方面， 通过使用在大肠杆菌中表达的不同衣壳蛋白（Cp）构建体， 模拟或重组天然样病毒颗粒。然而，这些结构不能正确地代表天然HBV 由于缺乏Cp的核酸结合结构域、病毒基因组和病毒酶，所有的 这是病毒复制所必需的。此外，缺少Cp的翻译后修饰也 阻碍了从哺乳动物中获得的生物医学数据与观察到的Cp结构之间的解释 细胞培养系统或实验动物。迄今为止，还没有可用的高分辨率天然HBV结构， 这是HBV领域知识的一个主要空白。 在这个建议中，我们的目标是使用冷冻电子显微镜（cryo-EM）直接表征的结构， 来自人细胞的天然HBV衣壳和病毒体。在目标1中，我们将确定 具有不同类型病毒基因组的纯化的细胞内HBV衣壳。我们将解决关键问题 关于HBV衣壳在基因组成熟过程中的结构动力学。我们还将确定 HBV逆转录酶（RT）的结构及其在逆转录过程中的位置，以帮助了解 其作用模式（RT是静态的还是移动的）。在目标2中，我们将研究高分辨率结构 分泌的HBV病毒体。这一目标将解决关于HBV衣壳如何与病毒相互作用的问题。 包膜蛋白质。最后，这两个目的的实验结果将被整合，以阐明衣壳蛋白 HBV复制过程中的动力学，并阐明HBV复制的分子决定因素。 这一提议有望解决4种类型的细胞内HBV衣壳结构（空的、RNA填充的、单- 双链DNA填充的和成熟的部分双链DNA填充的衣壳）和3个分泌的包膜HBV 病毒体结构（空的、成熟的和过早分泌的病毒体），使用cryo-EM确定构象 病毒复制过程中衣壳的变化，特别是在不同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Cp的适当突变 和RT，以确保获得如上所述的各种类型的均匀颗粒，其可以进一步 通过计算进行分类，以尽量减少交叉污染。 了解天然HBV结构将为HBV生物学提供有价值的新信息，并指导HBV的治疗。 未来新型抗病毒药物的设计。该项目预计将影响包括乙型肝炎病毒， 分子病毒学、抗病毒药物开发以及大分子结构和功能。