A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors

Abstract

In 2024, several human infections with highly pathogenic clade 2.3.4.4b bovine influenza H5N1 viruses in the United States raised concerns about their capability for bovine-to-human or even human-to-human transmission. In this study, analysis of the hemagglutinin (HA) from the first-reported human-infecting bovine H5N1 virus (A/Texas/37/2024, Texas) revealed avian-type receptor binding preference. Notably, a Gln²²⁶Leu substitution switched Texas HA binding specificity to human-type receptors, which was enhanced when combined with an Asn²²⁴Lys mutation. Crystal structures of the Texas HA with avian receptor analog LSTa and its Gln²²⁶Leu mutant with human receptor analog LSTc elucidated the structural basis for this preferential receptor recognition. These findings highlight the need for continuous surveillance of emerging mutations in avian and bovine clade 2.3.4.4b H5N1 viruses.

Fig. 1.. Receptor characterization of wild-type bovine Texas H5 HA from A/Texas/37/2024 (H5N1).

(A) The crystal structure of Apo Texas H5 HA was determined at 2.70 Å resolution (table S1). One HA monomer is highlighted in yellow for HA1 and green for HA2. The receptor binding site is composed of the 130-loop, 150-loop, 190-helix, and 220-loop (see inset). Key residues and additional mutations analyzed in this study are shown as red and lavender dots. (B) Sequence comparison of 190-helix and 220-loop of WT-Texas with human pandemic H1, H2 and H3 HA, and human-infecting H5N1 HA. The hallmark residues for switch of receptor binding preference in H1, H2 and H3 are colored with red dots. (C) Binding affinity was measured by surface plasmon resonance (SPR). Response units are plotted against protein concentration. The binding curves representing α2–3 and α2–6 are represented as blue and red, respectively. (D) The α2–3 and α2–6 sialosides in the ELISA are based on linear (left panel, open shapes) and biantennary (right panel, solid shapes) N-linked glycans, each with 1–3 repeats of LacNAc (Galβ1–4GlcNAc), as shown in the diagrams below. L: Linear. N: N-linked. LN₃-L, a non-sialylated glycan. Abs: Absorbance. R: -LCLC-Biotin. (E) Glycan microarray analysis of wild-type bovine Texas H5 HA. Two subsets of sialosides for α2–3 (glycan 3 to 26, black bar) and α2–6 (glycan 27 to 46, white bar) sialylated glycans imprinted on the chip are listed in table S2. RFU: relative fluorescence units.

Fig. 2.. Receptor specificity of bovine Texas H5 HA with a Q226L mutation and in combination with other mutations.

The glycan receptor specificity for the H5 HA with Leu226, Leu226/Ser228, Lys224/Leu226, and Lys224/Leu226/Ser228 mutations in the RBS were measured by SPR (A), ELISA (B) and glycan array (C). A/California/04/09 (CA04) H1 HA was used as a positive control for binding to α2–6 sialosides. (A) The K_D for each mutant in SPR assays was calculated based on the response units in figs. S1 and S2 using the Biacore S200 evaluation software static affinity model. (B) Extended linear (left panel) and biantennary (right panel) α2–3 and α2–6 sialosides were used in the ELISA. Abs: Absorbance. The schematic structures of the glycans are represented in Fig. 1. (C) Texas HA Leu226 mutant binding to the glycan array. Glycan numbers 3 to 26 on the array are for α2–3 sialosides (black bars) and 27 to 46 for α2–6 sialosides (white bars). The schematic shows main glycan structures represented in the binding to α2–6 sialosides. RFU: relative fluorescence units. Glycan numbers refer to the glycans illustrated in table S2.

Fig. 3.. Crystal structure of bovine Texas H5 HA with avian receptor analog LSTa and comparison with other H5 HA structures bound to LSTa.

(A) Receptor binding site (RBS) of WT Texas H5 HA (yellow carbon, blue nitrogen and red oxygen atoms) bound with LSTa (cyan carbon, blue nitrogen and red oxygen atoms). (B) RBS of VN1194 H5 HA bound with LSTa (PDB code 3ZP0). (C) RBS of Indo05 H5 HA bound with LSTa (PDB code 4K63). (D) Superposition of LSTa in Texas H5 HA and VN1194 H5 HA. (E) Superposition of LSTa in Texas H5 HA and Indo05 H5 HA. The HA structures are in a similar orientation after superimposition of their receptor binding subdomain (residues 117–265).

Fig. 4.. Crystal structure of bovine Texas H5 HA Leu226 mutant with human receptor analog LSTc and comparison with other HA structures bound to LSTc.

(A) Receptor binding site (RBS) of Texas H5 HA Leu226 mutant (yellow carbon, blue nitrogen and red oxygen atoms) bound to LSTc (cyan carbon, blue nitrogen and red oxygen atoms). (B) RBS of Viet04 H5 HA ferret-transmissible mutant (N158D/N224K/Q226L/T318I, H3 numbering) with LSTc (PDB code 4KDO). (C) RBS of Indo05 H5 HA airborne ferret-transmissible mutant (H110Y/T160A/Q226L/G228S, H3 numbering) with LSTc (PDB code 4K67). (D) RBS of dkEgy10 H5 HA human receptor preference E5.1 mutant (Q226L) with LSTc (PDB code 5E30). (E) RBS of 57H2 HA with LSTc (PDB code 2WR7). (F) Superposition of LSTc in Texas HA Leu226 mutant and in a Viet04 H5 HA ferret-transmissible mutant. (G) Superposition of LSTc in Texas HA Leu226 mutant and in the Indo H5 HA ferret-transmissible mutant. (H) Superposition of LSTc in Texas HA Leu226 mutant and dkEgy10 H5 HA human receptor preferring mutant. (I) Superposition of LSTc in Texas HA Leu226 mutant and 57H2 HA. The HA structures are in a similar orientation after superimposition of their receptor binding subdomain (residues 117–265).