X-ray Diffraction Analysis of Human Adenovirus

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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.