Structure And Assembly Of The Hepatitis B Nucleocapsid Protein

乙型肝炎核衣壳蛋白的结构和组装

• 批准号: 8746496
• 负责人: PAUL T WINGFIELD
• 金额: $ 79.62万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746496
• 关键词: Acute Liver Failure; Affinity; Antibodies; Antigen-Antibody Complex; Antigens; B-Lymphocytes; Binding; Biological; Biological Assay; C-terminal; Cancer Etiology; Capsid; Childhood; Chronic; Chronic Hepatitis B; Clinical; Clinical Markers; Code; Complex; Cryoelectron Microscopy; Cytoplasm; Deposition; Docking; Drug Design; Drug Targeting; Encapsulated; Escherichia coli; Genes; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Virus; Hepatitis B e Antigens; Human; Immune response; Immune system; Infection; Intervention; Life Cycle Stages; Maintenance; Metabolic; Modeling; Molecular; Molecular Conformation; Molecular Structure; Monoclonal Antibodies; Mus; N-terminal; National Institute of Allergy and Infectious Disease; Nucleocapsid; Nucleocapsid Proteins; Nucleosome Core Particle; Patients; Phage Display; Play; Positioning Attribute; Process; Protamines; Protein Precursors; Proteins; Publishing; RNA; RNA-Directed DNA Polymerase; Recombinants; Role; Sampling; Structure; T-Lymphocyte; Technology; Time; Tissues; Vaccines; Viral Proteins; Virus; Virus Diseases; Work; X-Ray Crystallography; base; dimer; hepatitis B virus P protein; humanized antibody; liver injury; prevent; small molecule; virus core

HBcAg (residues1-183) has been expressed in E.coli where it assembles in the bacterial cytoplasm into icosahedral capsids. Deletion of the polybasic C-terminal 34 residues (protamine domain) produces assembly competent protein (residues 1- 149) which is suitable for structural analysis. The structure of the capsids has been previously determined by cryo-electron microscopy and by X-ray crystallography. The closely related HBeAg is a soluble secreted protein which is thought to modulate both the innate and adaptive immune responses so as to favor persistent or chronic infection. HBeAg is also an important clinical marker of HBV infection. HBeAg (159 residues) is truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein. An HBeAg-specific Fab was found to form a stable complex with the recombinant produced HBeAg protein. The immune complex was crystallized and the structure of HBeAg was determined for the first time and published Jan 2013. This structure precludes capsid assembly and forms a distinct antigenic repertoire, explaining why HBcAg and HBeAg are cross-reactive at the T cell level (through sequence identity) but not at the B cell level (through conformation). HBeAg is thought to regulate immune responses by direct interaction with proteins, for example Mal, which regulate the innate immune system. Using the structure of HBeAg we have been modeling its interaction (docking) with target proteins as a guide for new structural studies. The direct targeting of HBeAg is also a potential for the treatment of chronic HBV, and to assist in these studies we have used phage display technology to generate a panel of high-affinity humanized antibodies against HBeAg. Biophysical and structural studies are being carried out to screen these antibodies, with the aim of generating small molecules which bind and inhibit HBeAg function. For clinical use, we also are using the characterized antibodies to develop a highly specific assay for the HBeAg. Previous structural determinations of nucleocapsid-antibody immune complexes by cryo-electron microscopy were performed with a panel of murine antibodies. This work was extended to include human antibodies from clinical samples and a specific antibody (E1) binding to HBcAg capsids which may contribute to acute liver failure (ALF). The monoclonal antibody E1 Fab was generated from previous work (R.H. Purcell, NIAID) based on immunological and molecular biological studies of tissue from patients with ALF. The E1 Fab binds with high affinity to HBcAg core particles. The massive deposit of immune complexes leads to the liver damage associated with ALF. We are currently attempting to co-crystallize the E1 with soluble capsids dimers to determine the structure of the immune complex. This information may be useful for designing drugs which block the E1-capsid interaction, hence preventing or modulating ALF.

HBcAg（残基1 -183）已在大肠杆菌中表达，在细菌细胞质中组装成二十面体衣壳。多元C-末端34个残基（鱼精蛋白结构域）的缺失产生适合于结构分析的有装配能力的蛋白（残基1- 149）。衣壳的结构先前已经通过冷冻电子显微镜和X射线晶体学确定。与之密切相关的HBeAg是一种可溶性分泌蛋白，被认为可调节先天性和适应性免疫应答，从而有利于持续或慢性感染。HBeAg也是HBV感染的重要临床标志物。HBeAg（159个残基）在第149位截短，此外还含有来源于前体蛋白部分加工的10个残基N-末端延伸。发现HBeAg特异性Fab与重组产生的HBeAg蛋白形成稳定的复合物。免疫复合物被结晶，HBeAg的结构首次被确定，并于2013年1月发表。这种结构排除了衣壳装配并形成了独特的抗原库，解释了为什么HBcAg和HBeAg在T细胞水平上（通过序列同一性）而不是在B细胞水平上（通过构象）具有交叉反应性。HBeAg被认为通过与蛋白质（例如Mal）直接相互作用来调节免疫应答，Mal调节先天免疫系统。使用HBeAg的结构，我们一直在模拟其与靶蛋白的相互作用（对接），作为新结构研究的指导。HBeAg的直接靶向也是治疗慢性HBV的一种潜力，为了帮助这些研究，我们使用噬菌体展示技术来产生一组针对HBeAg的高亲和力人源化抗体。正在进行生物物理和结构研究以筛选这些抗体，目的是产生结合和抑制HBeAg功能的小分子。对于临床应用，我们还使用特征抗体开发针对HBeAg的高度特异性检测方法。 先前用一组鼠抗体通过冷冻电子显微镜进行核衣壳-抗体免疫复合物的结构测定。这项工作扩展到包括来自临床样本的人抗体和与可能导致急性肝衰竭（ALF）的HBcAg衣壳结合的特异性抗体（E1）。单克隆抗体El Fab由先前的工作产生（R.H.珀塞尔，NIAID）基于来自ALF患者的组织的免疫学和分子生物学研究。E1 Fab以高亲和力结合HBcAg核心颗粒。免疫复合物的大量存款导致与ALF相关的肝损害。我们目前正试图将E1与可溶性衣壳二聚体共结晶，以确定免疫复合物的结构。这些信息可能有助于设计阻断E1-衣壳相互作用的药物，从而预防或调节ALF。