Norwalk virus, belonging to the family of Caliciviridae, is the major cause of epidemic non-bacterial gastroenteritis in humans. Norwalk virus represents an emerging virus as increased clinical significance of these agents is being recognized as new methods to detect these viruses are being utilized. This virus, which is exclusively a human pathogen, is unique among human viruses as the capsid is formed by one structural protein. At least four antigenic types of these human pathogens have been identified and more probably exist. Studies of animal caliciviruses have identified multiple serotypes indicating it may be difficult to develop vaccines. Instead, other antiviral strategies need to be devised. Norwalk virus has not yet been cultivated in cell culture, but the cloning and expression of its genome resulted in the discovery that the capsid protein when expressed using the baculovirus system spontaneously assembles into virus-like particles. The three-dimensional structure of the baculovirus expressed Norwalk virus, determined using electron cryomicroscopy and computer image reconstruction at approximately 22 Angstrom resolution, exhibits T=3 icosahedral symmetry with a distinctive architecture that includes 90 arch-like capsomeres surrounding 32 large hollows. We have crystallized the baculovirus-expressed Norwalk virus capsid (approximately 380 Angstroms in diameter) into crystals suitable for high resolution X- ray crystallography. The crystals diffract to approximately 3 Angstroms using synchrotron radiation which will allow the first structural determination of a calicivirus capsid to near atomic resolution. We also have produced a full-length cDNA of the viral genome. Based on our recent structural, molecular biologic and biochemical studies that have characterized these viruses, we propose to develop antiviral strategies for these unique single-stranded RNA human pathogens.
The specific aims of our studies are to design antiviral strategies (l) to inhibit virus binding to cells, (2) to inhibit capsid disassembly (uncoating) and capsid assembly, and (3) to inhibit calicivirus replication with minimal cell toxicity. We will determine the three-dimensional structure of Norwalk virus capsids to atomic resolution using X-ray crystallographic techniques and antibody virus complexes using electron cryomicroscopy and computer image processing. The atomic resolution structure together with biochemical analyses will provide insight into the nature of chemical interactions that govern the assembly, uncoating, receptor recognition and antigen neutralization in the Norwalk virus and will be used to develop antivirals. Because of the unique features of the calicivirus structure, it is anticipated that new types of antivirals will be designed. Expression systems of full-length and subgenomic RNAs also will be established in mammalian cell systems to obtain infectious particles and permit quantitative and toxicity testing of developed antivirals.
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