Infectivity studies of influenza virus hemagglutinin receptor binding site mutants in mice

Abstract

The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.

FIG. 1.

Left panel, ribbon diagram showing the HA trimer. One of the three monomers is colored to show the HA1 subunit in blue and the HA2 subunit in red. The region encompassing the receptor binding domain is shaded, and the location of bound sialic acid is indicated in green. The location of HA1 Tyr-98 at the base of the binding pocket is also indicated, as are some of the positions identified in reversion mutants that may influence trimer stability. Right panel, enlarged view of the receptor binding region. The binding site is a shallow depression bordered by a short α-helix (the 190 helix) at the membrane-distal edge, the 130 loop at the front of the site, and the 220 loop at the left side. Conserved residues Y98, W153, H183, and Y195 form the base of the site, and the positions of Y98 and W153 are indicated. Sialic acid is shown in green, and the location of residues identified in reversion mutants are indicated (as they appear in WT HA). The sialic acid binding pocket is located in the monomer colored in blue, but residues from the adjacent monomer (shown in gray) can influence binding, particularly if carbohydrate attachments are involved. This is illustrated by the location of the carbohydrate chain originating from HA1 Asn-165 of the neighboring monomer, shown here in gray and red at the left side of the binding site.

FIG. 2.

Time course of weight loss in mice infected with WT or Y98F mutant viruses.