MOLECULAR-BASIS FOR THE RESISTANCE OF INFLUENZA-VIRUSES TO 4-GUANIDINO-NEU5AC2EN

We report the selection and characterization of influenza A/NWS-G70c a nd B/HK/8/73 (HG) viruses which are resistant to the potent influenza neuraminidase inhibitor, 4-guanidino-Neu5Ac2en. Viruses were selected which replicated in MDCK cells in the presence of 20 mu g/ml inhibitor . The neuraminidase of resistant viruses was >200-fold more resistant to 4-guanidino-Neu5Ac2en than was the neuraminidase of the parent viru ses. Although amounts of neuraminidase protein were similar in resista nt and parent viruses, the enzyme activity of the resistant neuraminid ase heads was reduced by >95% for the substrates used. Relative to par ent viruses, the resistant viruses replicated to equal or greater tite rs in tissue culture and in embryonated chicken eggs. Sequence analysi s revealed a single nucleotide mutation in the neuraminidase gene of e ach virus resulting in the change of the conserved Glu 119 (which lies in a pocket beneath the active site of the enzyme) to Gly thus elimin ating an electrostatic interaction with the C-4 guanidinium moiety of the inhibitor. Mutations (Asn --> Ser) at amino acids 145 and 150 were also found in the hemagglutinin gene of the B/HK/8/73 (HG) virus resi stant to 4-guanidino-Neu5Ac2en. No changes were found in the hemagglut inin gene of the resistant A/NWS-G70c virus. (C) 1995 Academic Press, Inc.