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（由适用提供）：腺病毒（AD）是呼吸道，眼和胃肠道疾病的主要休闲药。在大量的人类基因转移试验以及抗菌疫苗的发展中，复制缺陷和有条件复制的AD载体也被采用。我们提出的研究利用了最新的进度和技术/专业知识在确定人类腺病毒的第一个晶体结构时获得的技术/专业知识。即使获得了显着的结构见解，但在精制X射线图与主要衣壳蛋白（己酮）的高分辨率冷冻结构和辅助蛋白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PVI的详细知识，该位置在细胞进入过程中在膜渗透中实现。我们建议在水泥蛋白的结构和身份方面解决差异，阐明通过使用MET/CYS的繁重原子标记保留和/或将病毒电子密度映射量的密集的原子标记来确定的独立蛋白，从而稳定了AD CAPSID的关键蛋白质 - 蛋白质相互作用，从而稳定了AD CAPSID。此外，我们将通过进行诱变和感染研究来评估变化的AD CAPSID的结构功能关系。具体而言，该提案将通过以下方式完成上述目标：1）准确识别辅助蛋白和相关的蛋白质 - 蛋白质相互作用，从而稳定天然ADV CAPSID并评估影响顶点区域稳定性的条件； 2）分析水泥蛋白的Deletin突变体的结构 - 功能关系以及基于矢量的基因输送中修饰的AD CAPSID的有效性和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提供对稳定腺病毒颗粒和结构功能关系的相互作用的更深入的理解，而AD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）是引起呼吸，眼和胃肠道疾病的大型且复杂的非发育病毒。目前，它们用作基因转移的向量的使用受到三维（3D）结构缺乏准确的知识的限制。拟议的研究旨在准确识别辅助蛋白及其蛋白质 - 蛋白质相互作用，这些蛋白质蛋白质相互作用，通过模型改进，沉重的原子标记研究以及参与结构功能研究，可以稳定AD CAPSID。