STRUCTURE AND ASSEMBLY OF THE HEPATITIS B NUCLEOCAPSID PROTEIN

背景乙型肝炎病毒(乙肝病毒)感染是一种 世界性的生物医学问题和对 病毒的组装和结构可能有助于开发新的 抗病毒疗法。乙肝病毒核心基因编码两种产物：(A) 形成核衣壳的183个残基核心抗原(HBcAg) 包裹病毒DNA的颗粒和多功能 乙肝病毒聚合酶；(B)前C蛋白(Pre-C)，它是N-和C-。 末端加工以形成分泌的非颗粒蛋白 称为e抗原(HBeAg)。HBcAg已在大肠杆菌中表达 它是否在细菌细胞质中组装成二十面体 衣壳，含有结合的宿主核酸。删除 多碱C-端34个残基也产生具有组装能力的 蛋白。C-末端截短蛋白(CPE：残基1-149) 不含核酸，可以高度提纯，而且更 比全长蛋白质更适合于结构研究。原生的 HBeAg也在第149位和In处C-末端截断 加合物含有来自以下来源的10个残基N-末端延伸 Pre-C部分处理。这种蛋白质和几个变种有 也在大肠杆菌中表达，用于结构研究。结果是 在早期的工作中，CPE的结构是由冷电子决定的 显微镜和图像分析，分辨率约为0.8纳米。 这使得能够辨别各种结构领域，尤其是 一个四个阿尔法螺旋丛，它形成了 衣壳。这些表面投影是通过标记来显示的 单抗是免疫显性的部位 表位，一个重要的血清学标志。本地化其他地区 功能和结构上的重要性，我们已经通过 不同位点活性半胱氨酸残基的定点突变 在可以用电子密度特殊标记的蛋白质中 试剂。我们之前定位了C-末端区域，一个 衣壳组装和形态发生的重要决定因素，以及 使用类似的方法正在继续映射其他功能 重要的区域，包括N-末端结构域，它也 似乎在组装过程中起到了作用。经过几年的连续 努力，为CPE的结晶创造了条件 出去。虽然去年CPE已经实现了结晶， 这些晶体很难复制，而且很小，而且 几个月后才能形成。此外，它们不能被冻结，a 使用同步加速器进行高分辨率研究的先决条件 消息来源。使用不同的纯化策略制备的衣壳可以 现在通常在不到一周的时间里从大到小 晶体(&lt；0.5 mm)，可转移防色剂 才能成功冷冻。意义和未来方向： 乙肝病毒衣壳的高分辨结构测定 X-射线衍射似乎可以使用由 已开发的方法。从低温电子导出的0.9 nm模型 显微复制将在结构工作中发挥主要作用，因为它提供了 一个合适的分子相变模型。乙肝病毒的其他目标 结构测定包括乙肝病毒聚合酶和HBeAg。 这些蛋白质或其功能区将在 进行了大肠杆菌和结构测定。总结 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，但慢性乙肝病毒通常是在 童年。乙肝病毒核衣壳在结构上起着重要的作用 病毒在生命周期中的作用和代谢作用。一个 对乙肝病毒核衣壳分子结构的认识 将允许有针对性的药物发现，目的是防止 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. The HBV core gene codes for two products: (a) the 183 residue core antigen (HBcAg) which forms nucleocapsid particles which encapsidate the viral DNA and a multifunctional HBV polymerase; (b) a precore protein (pre-C) which is N- and C- terminally processed to form a secreted non-particulate protein called e- antigen (HBeAg). 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 also produces assembly competent protein. The C-terminal truncated protein (Cpe: residues 1-149) does not contain nucleic acid, can be highly purified and is more suitable for structural studies than the full-length protein. 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. This protein and several variants have also been expressed in E.coli for structural studies. Results In earlier work, the structure of Cpe was determined by cryo- electron microscopy and image analysis to a resolution of about 0.8 nm. This allowed various structural domains to be discerned, especially a four alpha-helical bundle which formed the surface projections of the capsid. These surface projections were shown by labeling with monoclonal antibodies to be the site of the immunodominant epitope, an important serological marker. To localize other regions of functional and structural importance, we have introduced by site-directed mutagenesis reactive cysteine residues at various sites in the protein that can be specially labeled with electron dense reagents. We previously localized the C-terminal region, an important determinant of capsid assembly and morphogenesis, and using similar approaches are continuing to map other functionally important regions, including the N-terminal domain which also appears to play a role in assembly. After several years of continuous effort, conditions for the crystallization of Cpe have been worked out. Although crystallization of Cpe had been achieved last year, the crystals were difficult to reproduce, were small, and took several months to form. Furthermore, they could not be frozen, a prerequisite for high-resolution studies using a synchrotron light source. Capsids prepared using a different purification strategy, can now be routinely crystallized in less than a week to from large crystals (<0.5 mm) and which can be transferred cyropreservants for successful freezing. Significance and future direction: The high-resolution structure determination of the HBV capsid using X-ray diffraction appears possible using crystals prepared using the methods developed. The 0.9 nm model derived from cryo- electron microcopy will play a major role in the structural work by providing a suitable molecular phasing model. Other targets of HBV for structural determinations include the HBV polymerase and HBeAg. These proteins, or functional regions thereof, will be expressed in E.coli and structure determinations carried out. 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.