Structure And Assembly Of The Hepatitis B Nucleocapsid Protein

HBcAg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，其中含有结合的宿主核酸。多碱基c端34残基（鱼精蛋白结构域）的缺失也产生装配能力蛋白。c端截断蛋白（Cp149）衣壳不含核酸，其结构已通过冷冻电子显微镜、图像分析和x射线晶体学测定。原生HBeAg的c端也在149位被截断，此外还含有前体蛋白（pre-C）部分加工产生的10个残基n端延伸。尽管对HBeAg的功能和结构的详细了解尚不清楚，但它作为一种血清学标志物具有临床重要性。使用表面等离子体共振（Biacore）测量抗体抗原相互作用，确定了一组针对HBV核衣壳蛋白的单克隆抗体（mab）的动力学亲和力图。单克隆抗体（小鼠来源）结合组装（HBcAg）和非组装形式的衣壳（HBeAg）被鉴定出来，新的组合用于临床诊断描述。先前通过冷冻电镜对核衣壳-抗体免疫复合物的结构测定有助于更清楚地解释组装的HBcAg和未组装的HBeAg抗原之间的免疫学区别。这项工作已经扩展到包括人类抗体，初步结果表明结合到衣壳表面的不同区域（表位），这是我们之前使用模型小鼠抗体发现的。这项工作，连同我们之前对衣壳的免疫复合物和来自幼稚b细胞的表面免疫球蛋白的抗体的描述，提供了抗体与与重要人类疾病相关的病毒蛋白系统相互作用的最完整的结构图之一。

HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues (protamine domain) also produces assembly competent protein. The capsids from C-terminal truncated protein (Cp149) do not contain nucleic acid and their structure has been previously determined by cryo-electron microscopy and image analysis and by X-ray crystallography. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of precursor protein (pre-C). Although detailed knowledge of the function and structure of HBeAg are unknown it has clinical importance as a serological marker. Using surface plasmon resonance (Biacore) to measure antibody antigen interactions, a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins was determined. Monoclonal antibodies (murine origin) binding to the assembled (HBcAg) and non-assembled forms of the capsids (HBeAg) were identified and new combinations useful for clinical diagnosis described. Previous structural determinations of nucleocapsid-antibody immune complexes by cryo-electron microscopy helped to more clearly explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen. This work has been extended to included human antibodies and the preliminary results indicate binding to distinct regions on the capsid surface (epitopes) which we had previously identified using the model murine antibodies. This work, together our previous description of an immune complex of capsids and an antibody representative of the surface immunoglobulin from naive B-cells, provide one of the most complete structural pictures of the interaction of antibodies with a viral protein system related to an important human disease. Using a rabbit antibody library, monoclonal antibodies (mAbs) were selected then humanized to produce chimeric mAb fragment antigen binding portions (Fab). Fabs against the HBV capsid proteins were selected for high affinity binding to the capsid subunits. The binding of the antibodies prevent the assembly of the capsids (a central functional and structural component of the HBV virus) and may with development provide useful anti-HBV reagents. Direct determination of the biophysical properties of the HBV capsid protein is limited due to the size of the multiprotein megadalton complex. In collaboration with Albert Heck (Utrecht University) macromolecular tandem and ion mobility mass spectrometry was used to study the stability and conformational diversity of HBV capsids. Very precise mass measurements were made and the exact molecular stoichiometries of HBV nucleocapsid complexes determined for the first time. This work was extended to include measurements of stability and elasticity of the capsids which resulted in the detection of conformational heterogeneity in the capsid population. Also, the stability of the capsids was addressed by the direct determination of the slow exchange of constituent subunits using isotopically labeled protein. The methods and approaches used have general applicability to the study of large molecular assemblies used in nano- and biotechnology