STUDIES OF THE BINDING-PROPERTIES OF INFLUENZA HEMAGGLUTININ RECEPTOR-SITE MUTANTS

Site-specific mutations have been made in the influenza hemagglutinin (HA) receptor binding site to assess the contribution of individual am ino acid residues to receptor recognition. Screening of mutant HAs, ex pressed using recombinant vaccinia virus-infected cells, for their abi lities to bind human erythrocytes indicated that substitutions involvi ng conserved residues Y98F, H183F, and L194A severely restricted bindi ng and that the substitution W153A prevented cell surface expression o f HA Mutation of residues E190 and S228 that are in positions to form hydrogen bonds with the 9-OH of sialic acid appeared to increase eryth rocyte binding slightly, as did the substitution G225R. Substitutions of other residues that are directly or indirectly involved in receptor binding, S136T, S136A, Y195F, G225D, and L226P, had intermediate effe cts on binding between these two extremes. Estimates of changes in rec eptor binding specificity based on inhibition of binding to erythrocyt es by nonimmune horse sera indicated that mutants G225R and L226P, unl ike wild-type HA, were not inhibited; Y195F and G225D mutants were, li ke wild type, inhibited; and erythrocyte binding by mutants S136A, S13 6T, E190A, and S228G was only partially inhibited. Viruses containing mutant HAs Y98F, S136T, G225D, and S228G that cover the range of eryth rocyte binding properties observed were also constructed by transfecti on. Ail four transfectant Viruses replicated in MDCK cells and embryon ated hens' eggs as efficiently as wild-type X-31 virus, although the Y 98F mutant virus was unable to agglutinate erythrocytes. Mutant MDCK c ells that have reduced levels of cell surface sialic acids were suscep tible to infection by S136T, G225D, and S228G transfectant viruses and by wild type but not by the Y98F transfectant virus. (C) 1998 Academi c Press.