X-ray Diffraction Analysis of Human Adenovirus

人腺病毒的 X 射线衍射分析

• 批准号: 7689983
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7689983
• 关键词: Adenovirus Vector; Adenovirus hexon capsid protein; Adenoviruses; Area; Capsid; Capsid Proteins; Cardiovascular Diseases; Cells; Chicago; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Data Set; Development; Disease; Endosomes; Engineering; Enteral; Fiber; Freezing; Gene Transfer; Genetic Crossing Over; Genome; Goals; Growth; Human; Human Adenoviruses; Knowledge; Location; Malignant Neoplasms; Maps; Mediating; Methods; Minor; Molecular; Mutation; Peptide Hydrolases; Phase; Play; Process; Production; Proteins; Resolution; Roentgen Rays; Role; Science; Serotyping; Source; Staging; Structure; Synchrotrons; Temperature; Vaccines; Viral; Virion; Virus; Virus Assembly; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; antimicrobial; base; computerized data processing; conditionally replicative adenovirus; electron density; flexibility; gene transfer vector; improved; insight; mutant; particle; penton base; public health relevance; respiratory; three dimensional structure; vaccine development

DESCRIPTION (provided by applicant): Adenoviruses (Ad) are major causative agents of respiratory, ocular and enteric diseases. Replication-defective and conditionally replicating Ad vectors are also being employed in ~25% of human gene transfer as well as for the development of anti-microbial vaccines. Further progress in these areas is currently hampered by the lack of accurate structural information on intact Ad particles. While there are crystal structures for several of the major Ad capsid proteins (hexon, penton base and fiber), we lack detailed knowledge of their association upon assembly into intact Ad particles. Moreover, the precise location and structure of 9 other capsid proteins as well as the organization of the Ad genome in the virion core are unknown. In particular, we lack structural information on a key capsid protein that mediates endosome disruption during virus entry (Wiethoff et al., 2005). Therefore, the major goal of this proposal is to determine the virion structure of human adenovirus at near atomic resolution using x-ray crystallography. The topology and fold of the major and minor capsid proteins and their arrangement in wildtype as well as in a hyperstable/non-infectious mutant Ad will be investigated. This information should increase the knowledge of the underlying interactions that influence virus assembly/disassembly and cell entry. Furthermore, detailed knowledge on the structure of human adenovirus may facilitate the re-engineering of adenoviral vectors for gene transfer and/or vaccine development. We have successfully produced crystals of human adenovirus that diffract to ~ 5A resolution and likely beyond based on recent (Aug. 07) data collected at GM/CA CAT at APS, Chicago. Processing of a recent dataset to 5.6A (~27% complete, Rsym: 0.30), obtained from the diffraction of fresh crystals employing improved freezing conditions as well as higher beam flux, indicated that the space group is P1 (a=852.6, b=856.6, c=865.4, a=60.3, ¿=60.4, ?=61.9) rather than C2 that was determined using previous lower resolution data. Thus, we believe that we have now crossed over a significant threshold that will enable us to determine the structure of adenovirus at a resolution higher than previously obtained by cryoEM at 6.9A. The knowledge and expertise acquired by analyzing the structure of Ad by x-ray diffraction may facilitate the structural analysis of even larger and more complex icosahedral viruses that are beginning to emerge (Wilson, W. et al. Science 309: 1090-1092, 2005). PUBLIC HEALTH RELEVANCE: Adenoviruses are complex non-enveloped viruses, which are being used as gene transfer vehicles to treat cardiovascular diseases and cancer. Their use as vectors for gene transfer is currently limited by the lack of accurate knowledge on the three dimensional (3D) structures of all the constituent proteins and their organization. The proposed studies are aimed at determining the crystal structure of the entire human adenovirus virion at near atomic resolution employing x-ray diffraction methods.

描述（由应用程序提供）：腺病毒（AD）是呼吸，眼和肠道疾病的主要结构药物。在人类基因转移的约25％以及抗菌疫苗的开发中，复制缺陷和有条件复制的AD载体也被采用。目前，由于缺乏完整的广告颗粒的精确结构信息，这些领域的进一步进展受到阻碍。虽然有几种主要的AD Capsid蛋白（己酮，Penton碱基和纤维）的晶体结构，但我们在组装成完整的AD颗粒时缺乏详细的了解。此外，尚不清楚其他9种其他衣壳蛋白的精确位置和结构以及在病毒核心中的AD基因组的组织。特别是，我们缺乏有关键盘蛋白的结构信息，该蛋白会介导病毒进入期间内体破坏（Wiethoff等，2005）。因此，该提案的主要目的是使用X射线晶体学在接近原子分辨率下确定人腺病毒的病毒结构。将研究主要和次要衣壳蛋白的拓扑和折叠及其在WildType以及它们在降低/非感染性突变体AD中的排列。这些信息应增加影响病毒组装/拆卸和细胞进入的潜在相互作用的知识。此外，有关人腺病毒结构的详细知识可能有助于重新设计腺病毒载体以进行基因转移和/或疫苗发育。我们成功地生产了人类腺病毒的晶体，该晶体衍射为〜5A分辨率，并且可能基于最近在芝加哥APS的GM/CA CAT收集的（8月07日）数据。 Processing of a recent dataset to 5.6A (~27% complete, Rsym: 0.30), obtained from the diffraction of fresh crystals employing improved freezing conditions as well as higher beam flux, indicated that the space group is P1 (a=852.6, b=856.6, c=865.4, a=60.3, ¿ =60.4, ?=61.9) rather than C2 that was determined using previous lower resolution 数据。这就是我们认为，我们现在已经越过了一个明显的阈值，这将使我们能够以比Cryoem先前在6.9a上获得的分辨率确定腺病毒的结构。通过分析X射线衍射获得的AD结构获得的知识和专业知识可能促进了开始出现的更大且更复杂的二十体病毒的结构分析（Wilson，W。Science 309：1090-1092，2005）。公共卫生相关性：腺病毒是复杂的非发育病毒，它们被用作治疗心血管疾病和癌症的基因转移车辆。目前，它们用作基因转移的向量的使用受到所有构造蛋白及其组织的三维（3D）结构的准确知识的限制。拟议的研究旨在通过采用X射线衍射方法在接近原子分辨率下确定整个人类腺病毒病毒的晶体结构。