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capsids。多巴斯C末端34个残基（精完全结构域）的缺失会产生适合结构分析的组装蛋白（残基1-149）。 Capsids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是一种可溶的分泌蛋白质，被认为可以调节先天和适应性免疫反应，从而有利于持续或慢性感染。 HBEAG也是HBV感染的重要临床标记。 HBEAG（159个残基）在位置149处被截断，此外包含10个残基N末端延伸，这些末端延伸源自前体蛋白的部分加工。发现HBEAG特异性FAB与重组产生的HBEAG蛋白形成稳定的复合物。免疫复合物是结晶的，并首次确定了HBEAG的结构，并于2013年1月发布。这种结构排除了衣壳组件并形成了独特的抗原曲目，解释了HBCAG和HBEAG为何在T细胞水平（通过序列身份）在B细胞水平上（通过B细胞级别）（通过综合群）（通过sequormations）进行了交叉反应性。人们认为HBEAG可以通过与蛋白质直接相互作用（例如MAL）来调节免疫反应，例如MAL，从而调节先天免疫系统。使用HBEAG的结构，我们一直在建模其与靶蛋白的相互作用（对接）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，为了帮助这些研究，我们使用噬菌体显示技术来生成一组针对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。为了临床使用，我们还使用特征性抗体为HBEAG开发高度特异性的测定法。 用一系列鼠抗体对核素 - 抗体免疫复合物的先前结构测定。这项工作扩展到包括临床样品中的人类抗体和与HBCAG衣壳结合的特定抗体（E1），这可能导致急性肝衰竭（ALF）。单克隆抗体E1 FAB是根据先前的作品（R.H. Purcell，Niaid）基于ALF患者的组织的免疫和分子生物学研究而产生的。 E1 Fab与HBCAG核心颗粒具有高亲和力结合。免疫复合物的大量沉积导致与ALF相关的肝损伤。我们目前正在尝试将E1与可溶性衣壳二聚体共结合，以确定免疫复合物的结构。该信息可能对于设计阻断E1 capsid相互作用的药物可能很有用，因此可以防止或调节ALF。