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.

性状（由申请人提供）：腺病毒（Ad）是呼吸道、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载体也用于约25%的人类基因转移以及抗微生物疫苗的开发。这些领域的进一步进展目前受到缺乏完整Ad颗粒的准确结构信息的阻碍。虽然有几个主要的Ad衣壳蛋白（六邻体，五邻体基地和纤维）的晶体结构，我们缺乏详细的知识，他们的协会组装成完整的Ad颗粒。此外，其他9种衣壳蛋白的精确位置和结构以及Ad基因组在病毒体核心中的组织是未知的。特别地，我们缺乏关于在病毒进入期间介导内体破坏的关键衣壳蛋白的结构信息（Wiethoff等人，2005年）。因此，本提案的主要目标是使用X射线晶体学以近原子分辨率确定人腺病毒的病毒体结构。将研究野生型以及超稳定/非感染性突变体Ad中主要和次要衣壳蛋白的拓扑结构和折叠及其排列。这些信息应该增加对影响病毒组装/拆卸和细胞进入的潜在相互作用的了解。此外，关于人腺病毒结构的详细知识可以促进用于基因转移和/或疫苗开发的腺病毒载体的再工程化。根据最近（2007年8月）在芝加哥APS的GM/CA CAT收集的数据，我们已经成功地制备了人腺病毒晶体，其分辨率达到~ 5A，并可能超过5A。将最近的数据集处理为5.6 A（约27%完成，Rsym：0.30），从采用改进的冷冻条件以及更高的光束通量的新鲜晶体的衍射中获得，表明空间群为P1（a=852.6，B=856.6，c=865.4，a=60.3，=60.4，？= 61.9），而不是使用先前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，这将使我们能够确定腺病毒的结构，其分辨率高于先前通过cryoEM在6.9A获得的分辨率。通过X射线衍射分析Ad的结构所获得的知识和专业知识可以促进开始出现的甚至更大和更复杂的二十面体病毒的结构分析（Wilson，W.等人Science 309：1090-1092，2005）。公共卫生相关性：腺病毒是一种复杂的无囊膜病毒，被用作基因转移载体来治疗心血管疾病和癌症。它们作为基因转移载体的用途目前受到缺乏对所有组成蛋白质的三维（3D）结构及其组织的准确知识的限制。拟议的研究旨在确定整个人类腺病毒病毒粒子的晶体结构，在近原子分辨率采用X射线衍射方法。