Structure And Assembly Of The Hepatitis B Nucleocapsid

乙型肝炎核衣壳的结构和组装

• 批准号: 6823097
• 负责人: PAUL T WINGFIELD
• 金额: --
• 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/6823097
• 关键词: Escherichia coli; X ray crystallography; cryoelectron microscopy; crystallization; hepatitis B antigens; hepatitis B virus group; image processing; monoclonal antibody; nucleocapsid; protein biosynthesis; protein folding; protein purification; protein structure; protein structure function; structural biology; surface plasmon resonance; virus assembly; virus envelope; virus protein

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. Structural studies on HBeAg are continuing using mutants with improved physical characteristics. Work is also progressing on the large HBV polymerase protein. In an incremental approach we have expressed various functional domains of polymerse which will be subjected to crystallization trails. 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 would allow targeted drug discovery with the aim of preventing the assembly and formation of the virus.

背景：丙型肝炎病毒（HBV）感染是一个全球生物医学问题，对病毒的组装和结构有了改进的理解，可能有助于开发新的抗病毒疗法，并提供有关复杂大分子结构的基本信息。 HBV核心基因代码前蛋白（PER-C）进行部分处理，以形成一种分泌的非参数蛋白质，称为E抗原（HBEAG）或完全加工以产生核心抗原（HBCAG）。 HBCAG是一种183个残留蛋白，可围绕RNA逆转录酶复合物（HBV聚合酶）围绕。 HBCAG已在大肠杆菌中表达，如果它在细菌细胞质中组装成含有结合的宿主核酸的二十面体c骨。多基质C末端34残基（精完全结构域）的缺失也会产生组装胜任蛋白。来自C末端截短蛋白（CP149：残基1-149）的衣壳不含核酸及其结构，由低温电子显微镜和图像分析以及X射线晶体学确定。天然HBEAG在149的位置也被c末端截断，此外，还包含10个残基N末端延伸，这些末端延伸源自前C的部分处理。尽管HBEAG的功能和结构尚不清楚，但它是重要的血清学标记。结果：使用表面等离子体共振（BIACORE）一个针对HBV Nucleocapsid蛋白的单克隆抗体（MAB）的动力学亲和力图显示了一系列结合亲和力。通过冷冻电子显微镜（A.Steven）进一步表征了一些HBV Nucleocapsid-抗体复合物。结果表明，不连续的表位比以前所描述的更多。这些发现有助于解释组装的HBCAG和未组装的HBEAG抗原之间的免疫学区别。 HBEAG的结构研究正在继续使用具有改善物理特征的突变体。大型HBV聚合酶蛋白的工作也在进行。在一种增量方法中，我们表达了聚合物的各种功能域，这些域将受到结晶径。摘要：丙型肝炎病毒（HBV）是癌症的主要原因。尽管有疫苗，但慢性HBV通常是在儿童时期获得的。 HBV NucleocapsID在病毒生命周期中起重要的结构作用和代谢作用。对HBV Nucleocapsid的分子结构的理解将允许靶向药物发现，以防止病毒的组装和形成。