Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus.

We have previously characterized an influenza A (H1N1) virus which has host-dependent growth and receptor binding properties and have shown that a mutation which removes an oligosaccharide from the tip of the hemagglutinin (HA) by changing Asn-129 to Asp permits this virus to grow to high titer in MDBK cells, (C. M. Deom, A. J. Caton, and I. T. Schulze, Proc. Natl. Acad. Sci. USA 83:3771-3775, 1986). We have now isolated monoclonal antibodies specific for the mutant HA and have used escape mutants to identify alterations in HA sequence which reduce virus yields from MDBK cells without reducing those from chicken embryo fibroblasts. Two types of escape mutants which grow equally well in chicken embryo fibroblasts were obtained. Those with the parent phenotype contain Asn at residue 129 and are glycosylated at that site. Those with the mutant phenotype are unchanged at residue 129 but have a Gly to Glu substitution at residue 158, which is close to residue 129 on the HA1 subunit. Binding assays with neoglycoproteins containing N-acetylneuraminic acid in either alpha 2,3 or alpha 2,6 linkage to galactose showed that the MDBK-synthesized oligosaccharides at Asn-129 reduce binding to both of these receptors, leaving the HA's preference for alpha 2,6 linkages unchanged. Glu at residue 158 greatly reduces binding to both receptors without reducing virus yields from MDBK cells. We conclude that changes in the receptor binding properties of the HA can result either from direct alteration of the HA protein by host cell glycosylation or from mutations in the HA gene and that these changes generate heterogeneity that can contribute to the survival of influenza A virus populations in nature.