X-ray diffraction analysis of human adenoviruses

描述(申请人提供)：腺病毒(Ad)是引起呼吸道、眼部和胃肠道疾病的主要病原体。复制缺陷和有条件复制的Ad载体也被用于大量的人类基因转移试验以及抗微生物疫苗的开发。我们建议的研究利用了最近在确定3.5？分辨率的人腺病毒第一晶体结构方面取得的进展和获得的技术/专业知识。尽管获得了重要的结构洞察，但在精制的X射线图和主要衣壳蛋白(六邻体)以及辅助蛋白的高分辨率低温EM结构之间发现了一些显著的差异。此外，我们仍然缺乏对关键的水泥蛋白PVI的位置的详细了解，PVI与细胞进入过程中的膜穿透有关。我们建议通过获得HAdV的精制模型，使用Met/Cys残基的重原子标记和/或通过对接到病毒电子密度图中独立在高分辨率下确定的辅助蛋白的模块结构域，来解决水泥蛋白结构和鉴定方面的差异，阐明稳定Ad衣壳的关键蛋白质-蛋白质相互作用。此外，我们将通过进行诱变和感染性研究来评估改变的Ad衣壳的结构与功能的关系。具体地说，这项建议将通过以下方式实现上述目标：1)准确鉴定稳定天然ADV衣壳的辅助蛋白和相关的蛋白质-蛋白质相互作用，并评估影响顶点区稳定性的条件；2)分析水泥蛋白Deletin突变体的结构-功能关系和修饰的Ad衣壳在载体基因传递中的有效性；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该可以更好地理解稳定腺病毒颗粒的相互作用以及改变的Ad类型的结构-功能关系，这些类型具有独特的生物物理和生化特性。 与公共卫生相关：腺病毒(Ad)是一种大而复杂的无包膜病毒，可引起呼吸道、眼部和胃肠道疾病。目前，由于缺乏对三维结构的准确了解，它们作为基因转移载体的使用受到限制。这些研究旨在通过模型改进、重原子标记研究以及伴随的结构-功能研究，准确地识别稳定Ad衣壳的辅助蛋白及其蛋白质-蛋白质相互作用。

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.