Structure and receptor binding preferences of recombinant hemagglutinins from avian and human H6 and H10 influenza A virus subtypes

Abstract

During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections.

Importance: Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses.

FIG 1

Structure-based sequence alignment of the amino acid sequences of avH6, huH6, avH10, and huH10 compared to the HA sequences from different subtypes: A(H1N1)pdm09, Washington/5/2011; A(H5N1), Vietnam/1203/2004; A(H9N2), Bangladesh/0994/2011; A(H3N2), Victoria/361/2011; and A(H7N9), Shanghai/2/2013. Seasonal H3 antigenic sites (66–69) are designated A through E.

FIG 2

A structural overview of HA. (A) One monomer HA of avH6 (green) and huH6 (pale green). The potential glycosylation sites are labeled. (B) One monomer HA of avH10 (blue) and huH10 (light blue). The potential glycosylation sites are labeled. (C) Expanded view of the HA RBS with its three structural elements comprising the binding site, the 130 loop, 190 helix, and 220 loop; conserved residues at the base of the RBS pocket are shown as sticks. The avH6 HA RBS (green) is shown overlapping equivalent structures from huH6 (pale green), avH10 (blue), huH10 (light blue), H1pdm09 (Washington/5/2011) (yellow), H3 (Finland/486/2004) (salmon), H5 (Vietnam/1203/2004) (orange), and H7 (Shanghai/2/2013) (pink). All structural figures were generated with MacPyMOL (70).

FIG 3

Glycan microarray analysis of recHAs. (A) avH6 HA; (B) huH6 HA; (C) avH10 HA; (D) huH10 HA. Colored bars highlight glycans that contain α2-3 SA (blue), 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 (yellow), and non-SA (gray). Error bars reflect the standard deviations of the signals from six independent replicates on the array. Structures of the numbered glycans are found in Table 2.

FIG 4

Binding of the receptor analogs to the H6 HAs. (A) Overlap of α2-3 ligand binding in the receptor binding site from avH6 HA (green) and huH6 HA (pale green). The different residues between avH6 HA and huH6 HA near the RBS are shown as sticks. (B) RBS of avH6 HA with LSTa in the pocket. Putative hydrogen bond interactions between the glycan and the HA residues were calculated by LIGPLOT (71) and are shown as dotted lines. (C) RBS of huH6 HA with 3SLN bound in the pocket. Putative hydrogen bond interactions between the glycan and the HA residues are shown as dotted lines. (D) RBS of huH6 HA with 6SLN bound in the pocket. Putative hydrogen bond interactions between the glycan and the HA residues are shown as dotted lines. The 2Fo-Fc electron density maps of receptor analogs contoured at 1.0 σ are shown as orange grids in panels B, C, and D.