Enhanced human receptor binding by H5 haemagglutinins

Abstract

Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between Asn-186 and Gln-226. In contrast, a double mutant, Δ133/Ile155Thr, isolated in Egypt has greater avidity for human receptor while retaining wild-type avidity for avian receptor. Despite these increases in human receptor binding, none of the mutants prefers human receptor, unlike aerosol transmissible H5N1 viruses. Nevertheless, mutants with high avidity for both human and avian receptors may be intermediates in the evolution of H5N1 viruses that could infect both humans and poultry.

Keywords: Avian influenza virus; Biolayer interferometry; H5N1 influenza virus; Haemagglutinin; Haemagglutinin crystal structure; Receptor binding; Receptor specificity.

Fig. 1

Estimates of the affinity and specificity of receptor binding by mutant H5 influenza viruses. Binding of sialylglycopolymers containing α-2,3-sialolactosamine (3SLN, avian receptor analogue, red) and sialylglycopolymers containing α-2,6-sialolactosamine (6SLN, human receptor analogue, blue) by VN1194 mutants (b–d, f–h) and tyTy mutants (j–l) was characterised by biolayer interferometry (BLI) as detailed before (Lin et al., 2012). For comparison, data for wild-type VN1194 (a) and tyTy (i) viruses and theoretical binding curves for an aerosol transmissible H5 mutant HA (e) (Xiong et al., 2013a) are included.

Fig. 2

Structures of the receptor binding sites of mutant and wild-type HAs in complex with human receptor analogue as determined by X-ray crystallography. Human receptors bound to HAs of the VN1194 Ser227Asn/Gln196Arg (b), Asn186Lys (c) and tyTy Δ133/Ile155Thr (e) mutants are shown. Four conserved structural elements of the site: 190-helix, 220-loop, 130-loop and 150-loop are in ribbon representation and are labelled. The locations of the amino acid substitutions, together with Gln-226 are indicated. The α-carbon atoms of Arg-196 and the site of deletion, Ala-133 (Δ133, amino-nitrogen of Gly-134), are shown as spheres. The sugar components of the receptor analogues are coloured sialic acid (Sia-1, yellow), galactose (Gal-2, blue) and N-acetylglucosamine (NAG-3, red). Human receptor complexes formed by the Ser227Asn/Gln196Arg (b) and Asn186Lys (c) mutant HAs show electron density for Sia-1 and Gal-2 (Supplementary Fig. 3a and b). The Δ133/Ile155Thr mutant HA–human receptor complex shows good electron density for Sia-1, Gal-2 and NAG-3 (Supplementary Fig. 3c). The directions of the α-2,6-glycosidic bonds are indicated by arrows. Note that all human receptors are bound in cis-conformation. For comparison, complexes of human receptors with wild-type VN1194 (a), wild-type tyTy (d), and aerosol transmissible-mutant HAs (f) described before (Xiong et al., 2013a) are shown.

Fig. 3

Structures of the receptor binding sites of mutant and wild-type HAs in complex with avian receptor analogue as determined by X-ray crystallography. Avian receptors bound to the VN1194 Ser227Asn/Gln196Arg (b), Asn186Lys (c) and tyTy Δ133/Ile155Thr (e) mutant HAs are shown. The images of the receptor binding sites are rotated through 60° about a vertical axis relative to Fig. 2. The structure of the Ser227Asn/Gln196Arg avian receptor complex shows density for Sia-1 and Gal-2 (Supplementary Fig. 3d), and the avian receptor complexes with Asn186Lys and Δ133/Ile155Thr mutant HAs show electron density for all 3 sugars of the bound receptor (Supplementary Fig. 3e and f). The structural elements and selected residues in the receptor binding sites are indicated as in Fig. 2. For comparison, avian receptors bound to wild-type VN1194 (a), wild-type tyTy (d), and aerosol transmissible-mutant (f) are shown. The directions of the α-2,3-glycosidic bonds are indicated by arrows. Note that the avian receptors bound to the VN1194 Ser227Asn/Gln196Arg mutant (b), the Asn186Lys (c) and the aerosol transmissible-mutant (f) HAs are in cis-conformation and the wild-type VN1194 (a) and tyTy (d) HAs and the tyTy Δ133/Ile155Thr mutant (e) HA bind avian receptors in a trans-conformation.

Fig. 4

Detailed comparison of wild-type and mutant VN1194 avian receptor complexes. (a) Superposition of unliganded (grey) and avian receptor complexes (green) with wild-type VN1194 in two orientations. The different positions of Gln-226 and Asn-186 upon avian receptor binding are indicated by arrows. (b) A magnified view of the wild-type HA-avian receptor complex shows the water mediated hydrogen bonds between Gln-226, Asn-186, and the avian receptor bound in a trans conformation. (c) and (d) Magnified views of the Ser227Asn/Gln196Arg and the Asn186Lys mutant HA complexes with avian receptors. Note that as a result of the point mutation (Asn186Lys) or the change of side-chain rotamer (Ser227Asn), the water mediated hydrogen bond between Gln-226 and Asn-186 is lost, Gln-226 is in a lower position, similar to that of Gln-226 in the un-liganded wild-type HA (shown as grey sticks), and both mutants bind avian receptor in a cis conformation.

Fig. 5

Detailed comparison of wild-type and mutant tyTy receptor complexes. (a) Superposition of the tyTy wild-type (green) and the Δ133/Ile155Thr double mutant (blue) human receptor complexes. The region outlined is magnified on the right side of the figure to show the main-chain structural differences between the wild-type and the double mutant HAs near residue 133. The two water molecules in the receptor complex of the Δ133/Ile155Thr double mutant are shown to be close to the hydrophobic surface (grey) formed by Ile-155 and Ala-133 in wild-type HA. (b) Superposition of the tyTy wild-type (green) and the Δ133/Ile155Thr double mutant (blue) HA-avian receptor complexes. As in (a) the wild-type and the double mutant HAs have differences in main-chain structure near residue 133. The two water molecules in the receptor complex of the Δ133/Ile155Thr double mutant are close to the hydrophobic surface formed by Ile-155 and Ala-133 in wild-type HA.