A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

Abstract

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.

FIG. 1.

(A) Release of hemoglobin measured by absorbance at 540 nm of untreated and resialylated CRBCs lysed after hemadsorption on 293T cells transfected with 3 μg each of plasmids expressing A/duck/Ukraine/1/63 HA, A/Moscow/10/99 HA, A/South Carolina/1/18 HA, A/New York/1/18 HA, or an HA containing a D190E mutation in the A/New York/1/18 HA (“avian” HA). Each bar represents the average of three separate experiments. Values are reported as percent maximal absorbance. CRBCs were resialylated with α2,3 or α2,6 sialic acid as described in the text. (B) Representative Western blot of 293T cells transfected with 3 μg pCAGGS 1918 HA constructs. The blot was probed with an anti-1918 HA monoclonal antibody (kindly provided by Tom Moran) and an anti-actin monoclonal antibody (Sigma). Total cellular protein of 5 μg, 2.5 μg, or 1.25 μg was loaded as indicated. Quantification of three Western blots was performed using the public domain NIH Image program, ImageJ. HA expression was normalized to actin. AB, absorbance; GFP, green fluorescent protein.

FIG. 2.

(A) Hemagglutination assay of virus-like particles (VLPs) using unmodified (left) and VCNA-treated CRBCs (right). In both panels the top row corresponds to two hemagglutination units and the bottom row is a 1:2 dilution. (B) Hemagglutination assay of virus-like particles (VLPs) using CRBCs. The left panel shows SAα2,3Gal-resialylated CRBCs hemagglutinate VLPs expressing the A/New York/1/18 and the 1918 “avian” HAs but not A/South Carolina/1/18 HA. The right panel shows SAα2,6Gal-resialylated CRBCs hemagglutinate VLPs expressing the A/South Carolina/1/18 and A/New York/1/18 HAs but not 1918 “avian” HA. The amount of VLPs expressing the respective HAs was normalized with untreated CRBCs. In both panels the top row corresponds to two hemagglutination units and the bottom row is a 1:2 dilution. Production of SAα2,3Gal CRBCs was carried out in a 10% suspension with 0.5 mU of α2,3-(N)-sialyltransferase (Calbiochem) using 1.5 mM CMP-sialic acid in 50 μl for 2 h at 37°C. Production of SAα2,6Gal CRBCs was carried out in a 10% suspension with 1.0 mU of α2,6-(N)-sialyltransferase using 1.5 mM CMP-sialic acid in 125 μl for 2 h at 37°C.