Structure And Assembly Of The Hepatitis B Nucleocapsid P

背景：乙肝病毒感染是一个世界性的生物医学问题，对病毒的组装和结构的了解有助于开发新的抗病毒药物，并提供关于复杂大分子结构的基本信息。乙肝病毒核心基因编码前C蛋白(Pre-C)，它要么被部分加工形成一种被称为e抗原(HBeAg)的分泌型非颗粒蛋白(HBeAg)，要么被完全加工以产生核心抗原(HBcAg)。HBcAg是一种183个残基的蛋白质，包裹在RNA-逆转录酶复合体(乙肝病毒聚合酶)周围。如果HBcAg在细菌细胞质中组装成含有结合宿主核酸的二十面体衣壳，那么它就在大肠杆菌中得到了表达。多碱C-末端34个残基(鱼精蛋白结构域)的缺失也产生具有组装能力的蛋白。C端截短蛋白(Cp149：残基1-149)的衣壳不含核酸，其结构由低温电子显微镜、图像分析和X射线结晶学确定。天然HBeAg也在第149位被C-末端截断，此外还含有来自前-C部分处理的10个残基N-末端延伸。虽然HBeAg的功能和结构尚不清楚，但它是一种重要的血清学标志。结果：进一步研究了核衣壳蛋白的组装：通过定点突变鉴定了影响组装的残基。在现有结构信息的基础上，对核心区(Cp149)进行了一系列突变。选择了似乎在衣壳亚基之间接触的残基。其中一个这样的突变用甘氨酸残基取代了丙氨酸残基，导致蛋白质亚基不能结合成衣壳。正在对未组装的衣壳进行研究，以对比组装后其免疫学、生化和结构特性的变化。连接鱼精蛋白结构域形成壳(核心区)的九个氨基酸序列在组装过程中也起着关键作用。高分辨电子显微镜显示该连接肽位于衣壳结构中。结构和生化证据表明，在结合的病毒核酸存在的情况下，接头有助于衣壳结构的组装。乙肝衣壳的免疫学特性对于临床诊断和监测感染过程具有重要意义。针对衣壳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抗与组装的衣壳和衣壳亚单位的结合。同时，正在确定与衣壳结合的选定抗体的结构(A·史蒂文)。主要表位的结构图谱将很快完成，还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：其他用于结构测定的乙肝病毒靶标包括乙肝病毒聚合酶和HBeAg，这些蛋白已在大肠杆菌中表达，并正在积极寻找。与甲型和乙型肝炎不同的是，目前还没有针对丙型肝炎的疫苗。在美国，丙型肝炎的感染正在达到流行的程度，导致慢性肝病。包装病毒基因组的病毒核衣壳是组装活性病毒的关键组成部分。这些衣壳是在细菌中产生的，如果它们自我组装成与病毒中发现的类似大小的颗粒。目前正在对衣壳进行结构研究。摘要：乙肝病毒是世界范围内导致癌症的主要原因。虽然有疫苗可用，但慢性乙肝通常是在儿童时期感染的。病毒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。了解乙肝病毒核衣壳的分子结构将有助于有针对性地发现药物，以防止病毒的组装和形成。

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.