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: 7137989
• 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/7137989
• 关键词: 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 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. Monoclonal antibodies binding to the assembled and non-assembled forms of the capsids were identified. 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 antigen A review article was published summarizing these findings. Biophysical and structural studies on HBeAg are continuing using mutants with improved biophysical characteristics, especially solubility. We are preparing immune complexes of HBeAg with monoclonal antibodies to enhance crystal formation and subsequent structural studies using X-ray crystallography. Work is also continuing on our attempts to produce the HBV polymerase protein. We have also initiated studies on the HBV protein X, a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The protein has been expressed in E.coli and we are currently attempting to crystallize it for detailed structural determination. The surface glycoprotein of the HBV virus has also been expressed in yeast cells for potential structural determination. 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 other essential viral proteins including the polymerase and the X protein 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.

背景：乙型肝炎病毒（HBV）感染是一个世界性的生物医学问题，加深对病毒组装和结构的了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 核心基因编码前核心蛋白 (pre-C)，该蛋白要么部分加工形成称为 e 抗原 (HBeAg) 的分泌型非颗粒蛋白，要么完全加工产生核心抗原 (HBcAg)。 HBcAg 是一种包含 183 个残基的蛋白质，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。删除多碱基 C 端 34 个残基（鱼精蛋白结构域）也会产生装配能力蛋白。 C 端截短蛋白（Cp149：残基 1-149）的衣壳不含核酸，其结构通过冷冻电子显微镜、图像分析和 X 射线晶体学测定。天然 HBeAg 也在第 149 位 C 端被截短，此外还包含源自前 C 部分加工的 10 个残基 N 端延伸。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。 结果：使用表面等离子共振 (Biacore)，一组针对 HBV 核衣壳蛋白的单克隆抗体 (mAb) 的动力学亲和力图揭示了一系列结合亲和力。鉴定了与衣壳的组装和非组装形式结合的单克隆抗体。通过冷冻电子显微镜进一步表征了一些 HBV 核衣壳-抗体复合物 (A. Steven)。结果显示，不连续表位的数量比之前描述的要多。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。已发表的一篇评论文章总结了这些发现。 HBeAg 的生物物理和结构研究正在继续使用具有改善的生物物理特性（尤其是溶解度）的突变体。我们正在制备 HBeAg 与单克隆抗体的免疫复合物，以增强晶体形成以及随后使用 X 射线晶体学的结构研究。我们还在继续尝试生产 HBV 聚合酶蛋白。我们还启动了对 HBV 蛋白 X 的研究，这是病毒的关键调节蛋白，处于 HBV 感染、复制、发病机制和可能的致癌作用的交叉点。该蛋白质已在大肠杆菌中表达，我们目前正在尝试将其结晶以进行详细的结构测定。 HBV 病毒的表面糖蛋白也在酵母细胞中表达，用于潜在的结构测定。 摘要：乙型肝炎病毒 (HBV) 是全球癌症的主要原因。尽管有疫苗，但慢性乙型肝炎通常是在儿童时期感染的。 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 核衣壳和其他重要病毒蛋白（包括聚合酶和 X 蛋白）的分子结构将有助于发现靶向药物，以防止病毒的组装和形成。更清楚地了解临床 HBV 标志物 HBcAg 和 HBeAg 之间的结构差异可能会产生更好的诊断工具。