X-ray Diffraction Analysis of Human Adenovirus

描述（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。有复制缺陷和有条件复制的Ad载体也被用于约25%的人类基因转移以及抗微生物疫苗的开发。由于缺乏完整Ad粒子的准确结构信息，这些领域的进一步进展目前受到阻碍。虽然有几种主要的Ad衣壳蛋白（hexon， penton碱和纤维）的晶体结构，但我们对它们在组装成完整Ad颗粒时的关联缺乏详细的了解。此外，其他9种衣壳蛋白的精确位置和结构以及Ad基因组在病毒粒子核心中的组织结构尚不清楚。特别是，我们缺乏在病毒进入过程中介导内核体破坏的关键衣壳蛋白的结构信息（Wiethoff et al., 2005）。因此，本研究的主要目标是利用x射线晶体学在近原子分辨率下确定人腺病毒的病毒粒子结构。将研究野生型和超稳定/非传染性突变Ad中主要和次要衣壳蛋白的拓扑结构和折叠及其排列。这些信息将增加对影响病毒组装/拆卸和细胞进入的潜在相互作用的认识。此外，对人类腺病毒结构的详细了解可能有助于重组腺病毒载体，用于基因转移和/或疫苗开发。根据最近（2007年8月）在芝加哥APS的GM/CA CAT收集的数据，我们已经成功地生产出了衍射至~ 5A分辨率的人腺病毒晶体，甚至可能更高。利用改进的冷冻条件和更高的束流通量对新鲜晶体衍射得到的最新数据集进行处理至5.6A(~27%完成，Rsym: 0.30)，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在6.9A低温下获得的分辨率更高的分辨率确定腺病毒的结构。通过x射线衍射分析Ad结构所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. et al.）。科学309:1090-1092,2005)。公共卫生相关性：腺病毒是复杂的非包膜病毒，被用作治疗心血管疾病和癌症的基因转移载体。由于缺乏对所有组成蛋白及其组织的三维（3D）结构的准确了解，它们作为基因转移载体的使用目前受到限制。本研究的目的是利用x射线衍射方法在近原子分辨率下确定整个人腺病毒病毒体的晶体结构。

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.