Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus

Abstract

Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in The Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutinin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild-type recombinant NL219 HA to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. The results suggest that the additional glycosylation sequon increases binding affinity to avian-type α2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens, which calls attention to the need for further studies to fully understand the unique properties of these viruses.

Fig 1

Structural overview of NL219 HA monomer. (A) One monomer is shown, with the HA1 chain colored in green and the HA2 chain in cyan. The locations of the glycosylation sites are labeled. (B) RBS of NL219 HA with the three structural elements comprising this binding site, the 210-loop, 120-loop, and the 180-helix, colored blue, yellow, and purple, respectively. Conserved residues are shown as sticks. (C) RBS overlap of NL219 (green), Tk02-H7 (magenta), NY107-H7 (gray), and human H3 (yellow). The extended 140-loop was labeled. The glycan at Asn123 is displayed as sticks and overlaid with a Fo-Fc map contoured at 2.5σ (blue grid). All structural figures were generated with Mac PyMOL (12).

Fig 2

Binding of the avian receptor analog to the NL219 RBS. (A) RBS of NL219 with 3′SLN into the pocket. Putative hydrogen bond interactions between the glycan and the HA residues, calculated by hbplus (31), are shown as black broken lines. (B) Overlap of α2-3 ligands binding in the receptor binding site from NL219 (green), human H1 (magenta), avian H2 (blue), avian H3 (orange), avian H5 (yellow), avian H7 (cyan), and swine H9 (gray).

Fig 3

Binding of the human receptor analog to the NL219 RBS. (A) RBS of NL219 with 6′SLN into the pocket. Putative hydrogen bond interactions between the glycan and the HA residues, calculated by hbplus (31), are shown as black broken lines. (B) Overlap of α2-6 ligands binding in the RBS from NL219 (green), human H1 (magenta), avian H2 (blue), avian H3 (orange), avian H5 (yellow), and swine H9 (gray).

Fig 4

Glycan microarray and kinetic binding analysis of wild-type and mutant NL219 and NL219ΔCHO recombinant HAs. To assess the effect of HA1 Asn123 glycosylation on receptor specificity, NL219 (A) and NL219ΔCHO (B) recombinant HAs were analyzed by glycan microarray. The binding kinetics of both proteins to specific biotinylated glycans (3′SLN-b, 3′SLNLN-b, 6′SLN-b, and 6′SLNLN-b) immobilized onto biosensors were also analyzed by BLI (C and D). Glycans on the microarray are grouped according to SA linkage: α2-3 SA (blue) and α2-6 SA (red), α2-6/α2-3 mixed SA (purple), N-glycolyl SA (green), α2-8 SA (brown), β2-6 and 9-O-acetyl SA (magenta), and non-SA (gray). Error bars in panels A and B reflect the standard error in the signal for six independent replicates on the array. The structures of each of the numbered glycans are found Table S1 in the supplemental material. Specific α2-3 glycan structures that were used in biosensor assays but are also represented on the microarray are indicated by red columns and an asterisk (*) for 3′SLN (glycans 19 and 20) and number sign (#) for 3′SLNLN (glycan 22).

Fig 5

Movement of Arg131 near the Asn123 glycosylation site. The RBS overlaps of NL219 (green) and Tk02-H7 (magenta) are shown in the diagram. Residues mentioned in the main text are labeled and shown as sticks. The major structural elements of the RBS are labeled.