Hemagglutinin receptor specificity and structural analyses of respiratory droplet-transmissible H5N1 viruses

Abstract

Two ferret-adapted H5N1 viruses capable of respiratory droplet transmission have been reported with mutations in the hemagglutinin receptor-binding site and stalk domains. Glycan microarray analysis reveals that both viruses exhibit a strong shift toward binding to "human-type" α2-6 sialosides but with notable differences in fine specificity. Crystal structure analysis further shows that the stalk mutation causes no obvious perturbation of the receptor-binding pocket, consistent with its impact on hemagglutinin stability without affecting receptor specificity.

FIG 1

Glycan microarray analyses of respiratory droplet-transmissible H5N1 virus. The receptor specificity of whole inactivated VN1203/7xCal/04/09 virus with WT and mutant H5 HAs was assessed using glycan microarrays. Glycan microarrays comprised nonsialylated controls (gray bars; glycan no. 1 and 2 on the x axis), α2-3-linked sialosides (white bars; 3 to 35 on the x axis), α2-6-linked sialosides (black bars; 36 to 55 on the x axis), and glycans with both α2-3- and α2-6-linked sialic acids (checkered bars; 56 and 57 on the x axis). The y axis represents relative fluorescence units (RFU). The data shown represent control human virus (A/Kawasaki/173/07 [H1N1]) (A), WT VN1203 virus (B), VN1203 N224K Q226L HA mutant virus (C), VN1203 N158D N224K Q226L HA mutant virus (D), VN1203 N158D N224K Q226L T318I HA mutant virus (E), and VN1203 T318I HA mutant virus (F). A complete list of glycan structures can be found in Table S1 in the supplemental material.

FIG 2

Specificity of respiratory droplet-transmissible recombinant VN1203 and INDO5 H5 hemagglutinins. (A to J) The receptor specificity of recombinant H5 HAs, representing a series of WT and receptor mutants of VN1203 (A, C, E, G, and I) and INDO5 (B, D, F, H, and J), was analyzed using glycan microarrays as described for Fig. 1. Results are shown for binding to selected glycans for the wild-type HAs for VN1203 (A) and INDO5 (B) and various mutants of each HA (C to J). (K) The key shows the structures of glycans selected for comparison. The data for the full array can be found in Fig. S1 in the supplemental material.

FIG 3

Crystal structures of VN1203 transmissible mutant HA (N158D, N224K, Q226L, and T318I) and its comparison with wild-type and VN1203 HA RBS mutant HA (N158D, N224K, and Q226L). (A) Overall structure of the VN1203 transmissible mutant HA. One HA protomer of the trimeric complex is colored with HA1 in pink and HA2 in cyan, with the other two protomers in gray. The four mutated HA1 residues are shown with their side chains (yellow carbon atoms, blue nitrogen atoms, and red oxygen atoms). The HA RBS is depicted in green. (B) Structural comparison of the RBSs of VN1203 transmissible mutant (with yellow side chains and pink C_α atoms) and wild-type VN1203 HA (PDB code 3GBM, with blue side chains and light blue Cα atoms). The RBS in the transmissible mutant is wider than in the wild-type HA, with slight movement of loop 220 and helix 190 by about 0.7 Å and 0.6 Å, respectively. Otherwise, the overall structures as well as key binding residues are virtually identical, with an overall Cα RMSD of 0.5 Å for the HA1 domains. (C) Structural comparison of RBSs of VN1203 transmissible mutant (with yellow side chains and pink Cα atoms) and VN1203 RBS mutant (with gray chains and Cα atoms). The RBS secondary structure elements loop 130, helix 190, and loop 220 as well as key residue side chains are virtually identical, with a Cα RMSD of 0.2 Å for HA1 domains. (D) Structural comparison of the transmissible mutant HA (with yellow side chains and pink Cα atoms) and the RBS mutant HA (with gray side chains and Cα atoms) around HA1 318. No obvious conformational changes were observed from the T318I mutation other than replacement of the side chain of 318. It appears that the T318 mutation would increase the hydrophobic interactions between HA1 and HA2 of each H5 protomer.

FIG 4

Stability conferred by the H107Y mutation in INDO5 HA-containing viruses. The stability of INDO5 and selected mutants was assessed after incubation of the virus over time at 50°C. (A) Hemagglutination of turkey erythrocytes with treated virus. (B) Viral growth of treated virus in MDCK cells. Results are representative of two independent analyses.