Molecular and Antigenic Analysis of Human Calicivirus

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 based on an increased clinical significance of these agents being recognized as new methods to detect these viruses are utilized. Recent studies have found these viruses cause almost all (greater than 95 percent) outbreaks of nonbacterial gastroenteritis in the United States. This virus, which is exclusively a human pathogen, has a capsid structure formed by 180 copies of a single protein (ORF 2). A second protein (ORF 3) present in small amounts has recently been identified in virions. Studies of animal caliciviruses have identified persistent infections and some animal viruses have recently been shown to be genetically related to human caliciviruses. Multiple genetic types of caliciviruses exist and some of these represent different 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 spontaneously assembles into virus-like particles (VLPs) when expressed using the baculovirus system. A high resolution 3.4 Angstrom units structure of the recombinant Norwalk virus VLPs, determined by X-ray crystallography, has shown that these particles exhibit T=3 icosahedral symmetry with a distinctive architecture that includes 90 arch-like capsomeres surrounding 32 large hollows. The atomic resolution structure together with biochemical analyses have provided insight into the nature of the chemical interactions that govern the assembly, disassembly, and receptor recognition, and allowed the formulation of testable hypotheses about mechanisms that may regulate these pathways. This grant application outlines experiments to continue to use structural, molecular, and biochemical approaches to understand the assembly, expression and antigenic properties of these unique singlestranded RNA human pathogens. The specific aims of the proposed studies are to continue (1) to dissect the molecular interactions that regulate capsid assembly and disassembly, (2) to understand the role of ORF 3 in genome encapsidation, and (3) to map antigenic and biologic domains on the virus capsid. Because of the unique features of the calicivirus structure, it is anticipated that the results obtained will provide the foundation needed to develop new types of antivirals. Expression systems of full-length and subgenomic RNAs also will be established in mammalian cell systems that may permit replication of infectious particles in cell culture.