Picornaviruses are attractive experimental models for basic studies on protein structure and function. The protein shell has particularly simple architecture, 60 subunits organized as 12 pentamers, and the atomic structure of the protein subunit is now known for three of them: human rhinovirus 14 (HRV14), type 1 poliovirus (PV1) and mengovirus. Using a panel of 35 neutralizing monoclonal antibodies and 62 escape mutant we have identified four neutralization sites on HRV 14. In a parallel study we have used 15 neutralizing monoclonal antibodies to identify three neutralization sites on the Sabin strain of PV1. We believe that we have picked up a correlation between the shape of the neutralization curve produced by an antibody and the location of its binding site on the virus surface. The results suggest that spanning distance between sites across a 2-fold symmetry axis may be responsible for the correlation. We will extend our studies to determine the strength of the correlation and will apply the antibodies and mutants developed in this work toward a systematic comparative study on mechanisms of picornavirus neutralization and assembly. Selected antibodies will be sent to Purdue University of crystallographic determination of the 3- dimensional structure of the binding site. We sill study a new class of drugs (WIN drugs) which neutralize picornaviruses by binding to a pocket inside VP1. We have identified three classes of drug-resistant mutants of HRV14; H- mutants (able to grow at high drug concentrations (6 Mug/ml); L- mutants (able to grow at low concentrations (at 0.3 Mug/ml but not at 6 Mug/ml); and D-mutants which depend upon the drug for growth. We will assemble a collection of independent mutant of each class and identify the responsible amino acid alteration. We will use these mutants and wild type virus to examine the effect of the drug on a variety of physical properties including pH stability, permeability to cesium ion and isoelectric A-B transition. Selected mutants will be analyzed by x-ray crystallography at Purdue University to determine the effect of the mutation on the 3-dimensional structure of the pocket.
