Structure & Assembly Of Hepatitis B Nucleocapsid Protein

摘要：背景：乙型肝炎病毒（HBV）感染是一个世界性的生物医学问题，提高对病毒组装和结构的了解可能有助于开发新的抗病毒治疗方法，并为复杂大分子结构提供基础信息。HBV核心基因编码前核蛋白（pre-C），前核蛋白经过部分加工形成称为e抗原（HBeAg）的分泌非颗粒蛋白，或经过完全加工产生核心抗原（HBcAg）。HBcAg是一种183残基蛋白，包裹在rna -逆转录酶复合物（HBV聚合酶）周围。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：残基1-149）衣壳不含核酸，其结构通过低温电子显微镜、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10个残基n端延伸，这是由于pre-C的部分加工而产生的。虽然HBeAg的功能和结构尚不清楚，但它是一个重要的血清学标志物。

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.