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，并在细菌细胞质中组装成二十面体衣壳。删除多碱基 C 端 34 个残基（鱼精蛋白结构域）可产生适合结构分析的装配能力蛋白（残基 1-149）。衣壳的结构先前已通过冷冻电子显微镜和 X 射线晶体学确定。密切相关的 HBeAg 是一种可溶性分泌蛋白，被认为可以调节先天性和适应性免疫反应，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 位被截短，此外还包含源自前体蛋白部分加工的 10 个残基 N 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。免疫复合物结晶，并且首次确定了 HBeAg 的结构，并于 2013 年 1 月发表。这种结构阻止衣壳组装并形成独特的抗原库，解释了为什么 HBcAg 和 HBeAg 在 T 细胞水平上存在交叉反应（通过序列身份），但不是在 B 细胞水平（通过构象）。 HBeAg 被认为通过与蛋白质（例如调节先天免疫系统的 Mal）直接相互作用来调节免疫反应。利用 HBeAg 的结构，我们对其与目标蛋白的相互作用（对接）进行了建模，作为新结构研究的指南。 HBeAg 的直接靶向也是治疗慢性 HBV 的潜力，为了协助这些研究，我们使用噬菌体展示技术生成了一组针对 HBeAg 的高亲和力人源化抗体。正在进行生物物理和结构研究来筛选这些抗体，目的是产生结合和抑制 HBeAg 功能的小分子。对于临床应用，我们还使用特征化的抗体来开发一种高度特异性的 HBeAg 检测方法。 先前通过冷冻电子显微镜对核衣壳-抗体免疫复合物进行结构测定是使用一组鼠抗体进行的。这项工作扩展到包括来自临床样本的人类抗体和与 HBcAg 衣壳结合的特异性抗体 (E1)，这可能导致急性肝衰竭 (ALF)。单克隆抗体 E1 Fab 是根据之前的工作（R.H. Purcell，NIAID）基于 ALF 患者组织的免疫学和分子生物学研究而产生的。 E1 Fab 以高亲和力与 HBcAg 核心颗粒结合。免疫复合物的大量沉积会导致与 ALF 相关的肝脏损伤。我们目前正在尝试将 E1 与可溶性衣壳二聚体共结晶，以确定免疫复合物的结构。该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。