Genetic and molecular analyses of spontaneous mutants of human rhinovirus 14 that are resistant to an antiviral compound

Spontaneous mutants of human rhinovirus 14 resistant to WIN 52084, an antiviral compound that inhibits attachment to cells, were isolated by selecting plaques that developed when wild-type virus was plated in the presence of high (2 micrograms/ml) or low (0.1 to 0.4 micrograms/ml) concentrations of the compound. Two classes of drug resistance were observed: a high-resistance (HR) class with a frequency of about 4 x 10(-5), and a low-resistance (LR) class with a 10- to 30-fold-higher frequency. The RNA genomes of 56 HR mutants and 13 LR mutants were sequenced in regions encoding the drug-binding site. The HR mutations mapped to only 2 of the 16 amino acid residues that form the walls of the drug-binding pocket. The side chains of these two residues point directly into the pocket and were invariably replaced by bulkier groups. These findings, and patterns of resistance to related WIN compounds, support the concept that HR mutations may hinder the entry or seating of drug within the binding pocket. In contrast, all of the LR mutations mapped to portions of the polypeptide chain near the canyon floor that move when the drug is inserted. Because several LR mutations partially reverse the attachment-inhibiting effect of WIN compounds, these mutants provide useful tools for studying the regions of the capsid structure involved in attachment. This paper shows that the method of escape mutant analysis, previously used to identify antibody binding sites on human rhinovirus 14, is also applicable to analysis of antiviral drug activity.