X-ray diffraction analysis of human adenoviruses

人腺病毒的 X 射线衍射分析

• 批准号: 8470519
• 负责人: VIJAY S REDDY
• 金额: $ 44.53万
• 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/8470519
• 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; public health relevance; 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.

描述（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。复制缺陷和有条件复制的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最近取得的进展和获得的技术/专业知识，以3.5¿分辨率确定人类腺病毒的第一个晶体结构。尽管获得了重要的结构见解，但在改进的x射线图和主要衣壳蛋白（hexon）以及辅助蛋白的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的位置的详细了解，PVI在细胞进入过程中涉及膜穿透。我们建议解决水泥蛋白结构和身份方面的差异，阐明稳定Ad衣壳的关键蛋白质-蛋白质相互作用，方法是获得一个精细的hav模型，使用Met/Cys残基的重原子标记和/或通过与病毒电子密度图的附件蛋白的模块结构域进行连接，以高分辨率独立确定。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。具体而言，本方案将通过以下方法实现上述目标：1)准确识别稳定AdV天然衣壳的辅助蛋白和相关蛋白-蛋白相互作用，并评估影响顶点区域稳定性的条件；2)分析水泥蛋白缺失蛋白突变体的结构-功能关系以及修饰的Ad衣壳在载体基因传递中的有效性；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将有助于更好地理解稳定腺病毒颗粒的相互作用以及具有独特生物物理和生化特征的改变Ad类型的结构-功能关系。