Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus

Abstract

Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission.

FIG 1

Binding specificity and affinity of the AN1_WT virus and AN1 viruses carrying substitutions to sialylglycopolymers in the receptor binding site. (A to E) Ligand binding curves representing binding of AN1_WT (A), AN1_L217Q (B), AN1_G219S (C), A/VN/1194/04 (D), and A/NL/213/03 (E) to α2,3-sialyllactosamine (3′SLN) (in red) and α2,6-sialyllactosamine (6′SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y = B_max × X/(K_d + X)]. The values correspond to the means ± standard deviations calculated from the results of two independent assays performed in duplicate. OD, optical density. (F) The values corresponding to the dissociation constant at equilibrium (K_d) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the K_d values for the AN1_WT virus to the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.

FIG 2

pH threshold of fusion and thermostability of AN1_WT HA and mutant HAs. (A) Syncytium formation in Vero cells expressing wild-type or mutant AN1 HA proteins after exposure to different levels of pH. Cells were fixed using 80% ice-cold acetone, washed, and stained using a 20% Giemsa solution. The black boxes represent the range of pH values at which fusion was detected microscopically. (B) HA protein stability as measured by the ability of viruses to agglutinate turkey red blood cells after incubation at 50°C for the indicated times (minutes). Colors indicate the HA titers upon treatment at various time points at 50°C as shown in the legend.

FIG 3

Cartoon representation of a model of the trimer structure of HA of AN1 at different positions. (A) HA of AN1 with the substitutions studied annotated. (B) Mutant N94K (cyan) is predicted to interact with E74 in HA2 of the neighboring monomer, but its presence results in the loss of an interaction between E74 and Q76. (C) Mutant A210E (shown as orange sticks), close to G219 (magenta), is predicted to interact with the side chains of both T156 and S237 on the neighboring monomer. (D) At position 58, there is a gap between the monomers due to the interaction of K58, kinking the peptide backbone. Substitution K58I could allow the backbone to straighten and form additional intermonomer interactions. Mutations were introduced using the program Andante into the crystal structure of the HA of A/Shanghai/2/2013 H7N9 (PDB code 4LN6). The sequence is identical to that of AN1.

FIG 4

Binding specificity and affinity of the AN1_WT virus and AN1 viruses carrying substitutions that affect HA stability to sialylglycopolymers. (A to F) Ligand binding curves representing binding of AN1_N94K (A), AN1_K58I (B), AN1_A210E (C), AN1_{G219S N94K} (D), AN1_{G219S K58I} (E), and AN1_{G219S A210E} (F) to α2,3-sialyllactosamine (3′SLN) (in red) and α2,6-sialyllactosamine (6′SLN) (in blue). Binding curves were fitted using a one-site nonlinear regression in GraphPad [Y = B_max × X/(K_d + X)]. The values correspond to the means ± standard deviations calculated from the results of two independent assays performed in duplicate. (G) The values corresponding to the dissociation constant at equilibrium (K_d) were calculated from the nonlinear regression curves as the concentration of sialylglycopolymers needed to achieve half-maximal binding at equilibrium. P values were calculated using an unpaired two-tailed t test comparing the K_d values of the AN1_WT virus or the AN1_G219S virus with the values determined for the mutant viruses. Statistical significance (P values under 0.05) is indicated by an asterisk.