Structure & Assembly Of Hepatitis B Nucleocapsid Protein

总结：背景资料：B型肝炎病毒（HBV）感染是一个世界性的生物医学问题，对病毒组装和结构的进一步了解可能有助于开发新的抗病毒治疗方法，并提供关于复杂大分子结构的基本信息。HBV核心基因编码前核心蛋白（pre-C），其部分加工形成称为e抗原（HBeAg）的分泌型非颗粒蛋白，或完全加工产生核心抗原（HBcAg）。HBcAg是围绕RNA-逆转录酶复合物（HBV聚合酶）的183个残基的蛋白质。HBcAg已在大肠杆菌中表达，其在细菌细胞质中组装成二十面体衣壳，其含有结合的宿主核酸。多碱基C-末端34个残基（鱼精蛋白结构域）的缺失也产生有组装能力的蛋白。来自C-末端截短蛋白质（Cp 149：残基1-149）的衣壳不含核酸，并且通过冷冻电子显微镜和图像分析以及通过X射线晶体学确定其结构。天然HBeAg也在位置149处被C-末端截短，并且另外含有源自前C部分加工的10个残基的N-末端延伸。尽管HBeAg的功能和结构尚不清楚，但它是重要的血清学标志物。 结果如下：使用表面等离子体共振（Biacore），一组抗HBV核衣壳蛋白的单克隆抗体（mAbs）的动力学亲和力图显示了一系列结合亲和力。一些HBV核帽-抗体复合物通过冷冻电子显微镜进一步表征（A. Steven）。结果显示，不连续表位的数量比以前描述的要多。这些发现有助于解释组装的HBcAg和未组装的HBeAg抗原之间的免疫学差异。HBeAg的生物物理和结构研究正在继续使用具有改进的物理特性，特别是溶解度的突变体。我们正在制备HBeAg和单克隆抗体的免疫复合物，以增强晶体形成和随后的结构研究，使用X射线晶体学。大的HBV聚合酶蛋白的研究也在进行中。研究了B型肝炎病毒聚合酶基因序列对其在大肠杆菌中表达的影响。大肠杆菌进行研究。终止密码子以外的DNA序列似乎对表达蛋白的总长度有影响。正在研究这是否是由于终止密码子的抑制、翻译过程中的某种移码或聚合酶的翻译后不稳定性。 摘要：B肝炎病毒（HBV）是全球主要的癌症原因。虽然有疫苗可供使用，但慢性HBV通常在儿童时期获得。HBV核衣壳在病毒的生命周期中起着重要的结构作用和代谢作用。了解HBV核衣壳和基本HBV聚合酶的分子结构将允许靶向药物发现，目的是防止病毒的组装和形成。更清楚地了解临床HBV标志物HBcAg和HBeAg之间的结构差异可能会导致更好的诊断工具。

Summary: Background: Hepatitis B Virus (HBV) infection is a worldwide biomedical problem and an improved understanding of the assembly and structure of the virus may help develop new antiviral therapies as well as provide basic information on the structure of complex macromolecules. The HBV core gene codes for precore protein (pre-C) which is either partially processed to form a secreted non-particulate protein called e-antigen (HBeAg) or fully processed to produce core antigen (HBcAg). HBcAg is a 183-residue protein that encapsidate around a RNA-reverse transcriptase complex (HBV polymerase). 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: residues 1-149) do not contain nucleic acid and their structure 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 pre-C. Although the function and structure of HBeAg are unclear it is an important serological marker. Results: using surface plasmon resonance (Biacore) a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins revealed a range of binding affinities. Some of the HBV nucleocapsid-antibody complexes were characterized further by cryo-electron microscopy (A. Steven). The results revealed a greater number of discontinuous epitopes than had been described previously. The findings help explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigens. Biophysical and structural studies on HBeAg are continuing using mutants with improved physical characteristics, especially solubility. We are preparing immune complexes of HBeAg and monoclonal antibodies to enhance crystal formation and subsequent structural studies using X-ray crystallography. Work is also progressing on the large HBV polymerase protein. The effect of the hepatitis B polymerase gene sequence on its expression in E. coli is being studied. DNA sequences beyond the stop codon seem to have an effect on the total length of the expressed protein. Investigation of whether this is due to a suppression of the stop codon, some sort of frame shift during translation, or post-translational instability of the polymerase is being investigated. Summary: The Hepatitis B Virus (HBV) is the major worldwide cause of cancer. Although a vaccine is available, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural role and metabolic role in the life cycle of the virus. An understanding of the molecular structure of the HBV nucleocapsid and the essential HBV polymerase would allow targeted drug discovery with the aim of preventing the assembly and formation of the virus. A clearer understanding of the structural differences between the clinical HBV markers: HBcAg and HBeAg could result in better diagnostic tools.