Structure And Assembly Of The Hepatitis B Nucleocapsid P

乙型肝炎核衣壳 P 的结构和组装

• 批准号: 6680169
• 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/6680169
• 关键词: Escherichia coli; X ray crystallography; cryoelectron microscopy; crystallization; hepatitis B antigens; hepatitis B virus group; image processing; monoclonal antibody; nuclear magnetic resonance spectroscopy; nucleocapsid; protein biosynthesis; protein folding; protein purification; protein structure; protein structure function; site directed mutagenesis; structural biology; 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: The assembly of the nucleocapsid protein has been further studied: residues which influence assembly has been identified by site-specific mutagenesis. A series of mutations were made in the core domain (Cp149) on the basis of available structural information. Residues which appear to be making contact between the subunits of the capsid were selected. One such mutation substituted a glycine residue for an alanine residue resulting in protein subunits which were unable to associate into capsids. Unassembled capsids are being studied to contrast the change in their immunological, biochemical and structural properties upon assembly. A nine amino acid sequence which links the shell forming (core domain) from the protamine domain also plays a key role in assembly. This linker peptide was located in the capsid structure by high resolution electron microscopy. The structural and biochemical evidence suggests the linker facilitates assembly of the capsid structure in the presence of bound viral nucleic acid. The immunological properties of Hepatitis B capsid are important clinically for diagnosis and monitoring the course of infection. Antibodies against various regions or epitopes of the capsid are also very useful tools for probing the structure. We are using biophysical techniques to measure the binding of various monoclonal antibodies to both assembled capsids and capsid subunits. In parallel, the structure of selected antibodies bound to capsids is being determined (A. Steven). The structural mapping of the major epitopes will soon be completed and will also provide basic information on the binding of antibodies to viral capsid structures. Significance and future direction: Other targets of HBV for structural determinations include the HBV polymerase and HBeAg these proteins have been expressed in E.coli and are being actively pursued. Unlike Hepatitis A and B, there is no vaccine against Hepatitis C. In the United States, Hepatitis C infection is reaching epidemic proportions, causing chronic liver diseases. The nucleocapsid of the virus which packages the viral genome is a critical component in the assembly of active virus. The capsids have been produced in bacteria were they self-assemble into particles of similar size to those found in the virus. Structural studies are being performed on the capsids. 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：核衣壳蛋白的组装已被进一步研究：影响组装的残基已通过位点特异性诱变鉴定。基于可用的结构信息，在核心结构域（Cp 149）中进行一系列突变。选择似乎在衣壳的亚基之间进行接触的残基。一个这样的突变用甘氨酸残基取代丙氨酸残基，导致蛋白质亚基不能结合成衣壳。正在研究未组装的衣壳，以对比组装后其免疫学、生物化学和结构特性的变化。从鱼精蛋白结构域连接壳形成（核心结构域）的九个氨基酸序列也在组装中起关键作用。通过高分辨率电子显微镜将该接头肽定位在衣壳结构中。结构和生物化学证据表明，接头在结合的病毒核酸存在下促进衣壳结构的组装。B型肝炎病毒衣壳的免疫学特性对临床诊断和监测感染过程具有重要意义。针对衣壳的不同区域或表位的抗体也是探测结构的非常有用的工具。我们正在使用生物物理技术来测量各种单克隆抗体与组装的衣壳和衣壳亚基的结合。与此同时，正在确定与衣壳结合的选定抗体的结构（A. Steven）。主要表位的结构图很快就会完成，也将提供抗体与病毒衣壳结构结合的基本信息。意义和未来方向：用于结构确定的HBV的其他靶点包括HBV聚合酶和HBeAg，这些蛋白质已在大肠杆菌中表达并正在积极研究中。与甲型肝炎和B型肝炎不同，目前还没有针对丙型肝炎的疫苗。在美国，丙型肝炎感染正在达到流行病的比例，导致慢性肝病。病毒核衣壳是病毒基因组的重要组成部分，是病毒组装的关键。这些衣壳是在细菌中产生的，它们自组装成与病毒中发现的颗粒大小相似的颗粒。正在对衣壳进行结构研究。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。