CHARACTERIZATION OF MUTANTS OF INFLUENZA-A VIRUS SELECTED WITH THE NEURAMINIDASE INHIBITOR 4-GUANIDINO-NEU5AC2EN

The development of viral resistance to the neuraminidase (NA) inhibito r, 4-guenidino-Neu5Ac2en, of influenza viruses was studied by serial p assage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidne y cells in the presence of increasing concentrations of inhibitor, Res istant mutants, selected after eight passages, had a 10,000-fold reduc tion in sensitivity to the inhibitor in plaque assays, but their affin ity (1/K-d) to the inhibitor was similar to that of the parental virus , Electron microscopic analysis revealed aggregation of the mutant vir us at the cell surface in the presence of the inhibitor, Sequence anal ysis established that a substitution had occurred in the NA (Arg-249 t o Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site, The cha nge at residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate cha nges in the hemagglutinin, After 13 passages of the parental virus, mu tants that were resistant to the high concentrations of inhibitor test ed were obtained, These viruses retained their drug-resistant phenotyp e even after five passages without the inhibitor, Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor, Sequence analysis of the NA ge ne from these drug-resistant mutants revealed an additional substituti on of Glu to Ala at the conserved amino acid residue 119, This substit ution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4 -guanidino-Neu5Ac2en is a multistep process requiring prolonged exposu re to the inhibitor.