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（由申请人提供）：腺病毒 (Ad) 是呼吸系统、眼部和肠道疾病的主要病原体，复制缺陷型和条件复制型 Ad 载体也被用于约 25% 的人类基因转移以及开发。目前，由于缺乏完整 Ad 颗粒的准确结构信息，尽管几种主要 Ad 衣壳蛋白（六邻体、五邻体）有晶体结构，但这些领域的进一步进展受到阻碍。碱基和纤维），我们缺乏它们在组装成完整 Ad 颗粒后的关联的详细知识，此外，其他 9 种衣壳蛋白的精确位置和结构以及病毒体核心中 Ad 基因组的组织也是未知的。我们缺乏在病毒进入过程中介导内体破坏的关键衣壳蛋白的结构信息（Wiethoff 等人，2005）因此，该提案的主要目标是使用近原子分辨率确定人类腺病毒的病毒体结构。 X 射线晶体学。将研究主要和次要衣壳蛋白的拓扑结构和折叠以及它们在野生型以及超稳定/非感染性突变体 Ad 中的排列。这些信息应该会增加对影响病毒的潜在相互作用的了解。此外，对人类腺病毒结构的详细了解可能有助于基因转移和/或疫苗开发的腺病毒载体的重新设计。根据最近（8 月 7 日）在芝加哥 APS 的 GM/CA CAT 收集的数据，将最近数据集处理至 5.6A（完成约 27%，Rsym：0.30），衍射至约 5A 分辨率，并可能更高。采用改进的冷冻条件以及更高的光束通量对新鲜晶体进行衍射，表明空间群为 P1（a=852.6，b=856.6， c=865.4, a=60.3, ¿ =60.4，?=61.9）而不是使用以前的较低分辨率数据确定的 C2 因此，我们相信我们现在已经跨越了一个重要的阈值，这将使我们能够以比以前获得的分辨率更高的分辨率确定腺病毒的结构。 6.9A 的冷冻电镜。通过 X 射线衍射分析 Ad 结构获得的知识和专业知识可能有助于对开始出现的更大、更复杂的二十面体病毒进行结构分析（Wilson，W.等人，Science 309：1090-1092，2005）。公共健康相关性：腺病毒是复杂的无包膜病毒，被用作治疗心血管疾病和癌症的基因转移载体，目前它们作为基因转移载体的用途有限。由于缺乏对所有组成蛋白质及其组织的三维 (3D) 结构的准确了解，拟议的研究旨在确定整个人类的晶体结构。采用 X 射线衍射方法以近原子分辨率分析腺病毒病毒颗粒。