Structure And Assembly Of The Hepatitis B Nucleocapsid Protein

HBcAg(残基1-183)已在大肠杆菌中表达，它在细菌细胞质中组装成二十面体衣壳。多碱C-末端34个残基(鱼精蛋白结构域)的缺失产生了适合结构分析的装配活性蛋白(残基1-149)。衣壳的结构以前已经通过冷冻电子显微镜和X射线结晶学确定。与HBeAg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和获得性免疫反应，从而有利于持续性或慢性感染。HBeAg也是乙肝病毒感染的重要临床标志物。HBeAg(159个残基)在第149位被截断，此外还含有一个来自前体蛋白部分加工的10个残基N-末端延伸。HBeAg特异性Fab与重组表达的HBeAg蛋白形成稳定的复合体。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组装并形成了一个独特的抗原库，解释了为什么HBcAg和HBeAg在T细胞水平(通过序列相同)而不在B细胞水平(通过构象)发生交叉反应。HBeAg被认为通过与蛋白质的直接相互作用来调节免疫反应，例如调节先天免疫系统的MAL。利用HBeAg的结构，我们一直在模拟它与目标蛋白的相互作用(对接)，作为新的结构研究的指南。直接靶向HBeAg也是治疗慢性乙肝的一种潜在方法，为了帮助这些研究，我们使用噬菌体展示技术来产生一组高亲和力的人源化抗体。目前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，我们还在使用这些特化的抗体来开发一种高度特异的HBeAg检测方法。 以前用冷冻电子显微镜测定核衣壳抗体免疫复合体的结构是用一组小鼠抗体进行的。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)，这可能导致急性肝功能衰竭(ALF)。单抗E1Fab是在免疫学和分子生物学研究的基础上从ALF患者的组织中产生的。E1Fab与HBcAg核心颗粒具有高亲和力。免疫复合体的大量沉积导致ALF相关的肝损伤。我们目前正试图将E1与可溶性衣壳二聚体共结晶，以确定免疫复合体的结构。这些信息可能有助于设计阻断E1-衣壳相互作用的药物，从而防止或调节ALF。

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.