X-ray diffraction analysis of human adenoviruses

人腺病毒的 X 射线衍射分析

• 批准号: 8292681
• 负责人: VIJAY S REDDY
• 金额: $ 47.38万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-22 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292681
• 关键词: Adenovirus Vector; Adenovirus hexon capsid protein; Adenoviruses; Affect; Antibodies; Antiviral Agents; Architecture; Binding; Biochemical; Capsid; Cell physiology; Cells; Characteristics; Complex; Cryoelectron Microscopy; Crystallization; DNA Packaging; DNA Sequence Rearrangement; Development; Docking; Effectiveness; Endosomes; Event; Fiber; Funding; Gastrointestinal Diseases; Gene Delivery; Gene Transfer; Genome; Goals; Human; Human Adenoviruses; Investigation; Knowledge; Label; Learning; Location; Macromolecular Complexes; Maps; Mediating; Membrane; Methods; Modeling; Molecular; Mutagenesis; Paper; Penetration; Proteins; Publishing; Reporting; Resolution; Role; Scaffolding Protein; Shapes; Staging; Structure; Structure-Activity Relationship; Surface; Technology; Vaccines; Viral; Virion; Virus; Virus Assembly; X ray diffraction analysis; X-Ray Diffraction; antimicrobial; base; cell assembly; comparative; conditionally replicative adenovirus; conformational alteration; design; electron density; gene transfer vector; improved; insight; meetings; mutant; novel; particle; protein protein interaction; receptor binding; respiratory; three dimensional structure; vector

DESCRIPTION (provided by applicant): Adenoviruses (Ad) are major causative agents of respiratory, ocular and gastrointestinal diseases. Replication-defective and conditionally replicating Ad vectors are also being employed in a significant number of human gene transfer trials as well as for the development of anti-microbial vaccines. Our proposed studies capitalize on the recent progress made and technology/expertise obtained in determining the first crystal structure of human adenovirus at 3.5 ¿ resolution. Even though significant structural insights were gained, a number of notable differences were found between the refined x-ray maps and the high-resolution cryoEM structures of the major capsid protein (hexon) as well as accessory proteins. Moreover, we still lack detailed knowledge of the location of the key cement protein, PVI, implicated in membrane penetration during cell entry. We propose to resolve the discrepancies with regards to the structure and identities of cement proteins, elucidate the critical protein-protein interactions that stabilize the Ad capsid by obtaining a refined model of HAdV, using heavy atom labeling of Met/Cys residues and/or by docking into the virus electron density maps the modular domains of accessory proteins, determined independently at high resolution. In addition, we will evaluate structure-function relationships of the altered Ad capsid by performing mutagenesis and infectivity studies. Specifically, this proposal will accomplish the above goals by: 1) accurately identifying the accessory proteins and the associated protein-protein interactions that stabilize the native AdV capsid and evaluate the conditions that affect the stability of the vertex region; 2) analyzing the structure-function relationships of the deletin mutants of cement proteins and the effectiveness of the modified Ad capsids in vector-based gene delivery and 3) determining the structure of immature AdV particles that represent an early stage of adenovirus assembly. These investigations and subsequent comparative analysis of their structures should provide greater understanding of the interactions that stabilize the adenovirus particle and structure-function relationships of the altered Ad types, which possess unique biophysical and biochemical characteristics. PUBLIC HEALTH RELEVANCE: Adenoviruses (Ad) are large and complex non-enveloped viruses that cause respiratory, ocular and gastrointestinal diseases. Their use as vectors for gene transfer is currently limited by the lack of accurate knowledge on the three dimensional (3D) structure. The proposed studies are aimed at accurately identifying the accessory proteins and their protein-protein interactions that stabilize the Ad capsid, by model refinement, heavy atom labeling studies as well as accompanying structure-function studies.

描述（由申请人提供）： 腺病毒（Ad）是呼吸道、眼部和胃肠道疾病的主要病原体。复制缺陷型和条件复制型 Ad 载体也被用于大量人类基因转移试验以及抗微生物疫苗的开发。我们提出的研究利用了在 3.5 分辨率下确定人类腺病毒第一个晶体结构方面所取得的最新进展和技术/专业知识。尽管获得了重要的结构见解，但在主要衣壳蛋白（六邻体）以及辅助蛋白的精细 X 射线图和高分辨率冷冻电镜结构之间发现了许多显着差异。此外，我们仍然缺乏对关键水泥蛋白 PVI 位置的详细了解，PVI 与细胞进入过程中的膜穿透有关。我们建议解决水泥蛋白结构和特性方面的差异，通过使用 Met/Cys 残基的重原子标记和/或通过对接到病毒电子密度图以高分辨率独立确定的辅助蛋白的模块化结构域，获得 HAdV 的精细模型，阐明稳定 Ad 衣壳的关键蛋白质-蛋白质相互作用。此外，我们将通过进行诱变和感染性研究来评估改变的Ad衣壳的结构-功能关系。具体来说，该提案将通过以下方式实现上述目标：1）准确识别辅助蛋白和相关的蛋白质-蛋白质相互作用，以稳定天然AdV衣壳并评估影响顶点区域稳定性的条件； 2) 分析水泥蛋白删除蛋白突变体的结构-功能关系以及修饰的 Ad 衣壳在基于载体的基因传递中的有效性，以及 3) 确定代表腺病毒组装早期阶段的未成熟 AdV 颗粒的结构。这些研究和随后对其结构的比较分析应该可以更好地理解稳定腺病毒颗粒的相互作用以及改变的Ad类型的结构-功能关系，这些类型具有独特的生物物理和生化特征。 公共卫生相关性：腺病毒 (Ad) 是一种大型且复杂的无包膜病毒，可引起呼吸道、眼部和胃肠道疾病。目前，由于缺乏对三维（3D）结构的准确了解，它们作为基因转移载体的使用受到限制。拟议的研究旨在通过模型细化、重原子标记研究以及伴随的结构功能研究，准确识别辅助蛋白及其稳定Ad衣壳的蛋白质-蛋白质相互作用。