SARS-CoV

Cloning, expression and biophysical analysis

An expression-ready clone library will be created and maintained containing all known and predicted coding regions for most avaliable strains. The expression-ready clone library will be constructed using experimentally validated clones for E. coli, baculovirus and mammalian expression systems and will be updated with any publicly released information. Sequencing of clone products will ensure quality assurance. Clones from the FSPS library will be used for protein production through primary expression strategies and predefined alternate pathways. Sufficient amounts of soluble folded proteins, domains, and other constructs will be expressed and purified for functional and structural analysis. Analytical and biophysical characterization approaches will be used to ensure high quality products.

NMR spectroscopy and X-ray crystallography

The structures and substructures of SARS-CoV proteins and complexes will be determined by using either NMR or X-ray crystallography. The method used will be based on size and protein behavior. Rapid optimization strategies through variation in clones, expression systems, and crystallization conditions will enhance the likelihood of successful structure determination. In addition, a set of pathways outside the traditional automated structure determination pipelines will be developed for more difficult structures, such as glycosylated and membrane-bound proteins. All structures will be fully validated, annotated, and released according to established standards of the NIH Protein Structure Initiative.

Viral life cycle, protein function, protein-protein interactions, and protein-ligand interaction

Several approaches will probe the function of each protein and its interactions, such as the characterization of the life cycle and host response; identification and characterization of protein-protein interactions, and indentification and characterization of ligands. Cryoelectron microscopy and iterative single-particle image reconstruction will be used to produce 2-D and 3-D maps of viral coat protein complexes from formalin-inactivated native and fusion-activated virus preparations. The effects on the viral lifecycle and intracellular host response for each SARS-CoV protein will be defined using a combination of viral cDNA cloning, site-directed mutagenesis, antisense functional mapping and microarray-based functional mapping using live virus produced from cDNA knockouts. In addition, SARS-CoV cellular receptors and the entry process will be identified and characterized using ligand "fishing" techniques.

Proteome map from experimental data

FSPS will collect and integrate existing data to derive hypotheses that will be experimentally tested utilizing the methods in sample production, functional proteomics, and structural proteomics. These bioinformatics services will support the pipeline of activities from design of primers to structural analysis. This will be done through sequence analysis that facilitates the in silico discovery phase of functional proteins, protein domains and substrates/cofactors/inhibitors, and large scale virtual docking to identify possible inhibitors of solved structures. Bioinformatics will additionally be used to reconstruct essential pathways and important networks involving SARS-CoV proteins, produce a prioritized list of possible drug/vaccine targets, and interact with the public data resources to optimize dissemination of data to the public.