Structural and functional analysis of surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals

Abstract

In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (S. J. Anthony, J. A. St Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles, W. I. Lipkin, MBio 3:e00166-00112, 2012, http://dx.doi.org/10.1128/mBio.00166-12). Here, we present a detailed structural and biochemical analysis of the surface antigens of the virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission, our results indicate that the A(H3N8) virus currently poses a low risk to humans.

Importance: Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here, we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. The results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses as these zoonotic influenza viruses continue to circulate and adapt to new hosts.

FIG 1

Structure of seal HA. (A) Overall structure, with one monomer HA highlighted in green (HA1 domain) and cyan (HA2 domain). Potential glycosylation sites are labeled, and those sites/glycans that could be visualized in the structure are shown as sticks (magenta). (B) Expanded view of the seal HA RBS with its three structural elements comprising the binding site—the 130 loop, the helix, and the 220 loop—colored yellow, red, and purple. Conserved residues are shown as sticks. (C) Comparison of the seal HA RBS (green) with overlapping equivalent structures from avian (cyan) and human (magenta) H3 HAs. Residue differences are shown as sticks. Seal HA is shown in cartoon form, while 3-SLN and interacting HA residues are shown as sticks. All structural figures were generated with MacPyMol (83).

FIG 2

Sequence alignment of the amino acid sequence of seal11 HA with those of the avian A(H3N8) virus A/Duck/Ukraine/1/1963 (dkUkr63; PDB ID 1MQL) and two human HA sequences from A/Aichi/2/1968 (Aichi68; PDB ID 3HMG) and the seasonal H3 vaccine strain from A/Hong Kong/4443/2005 (HK05; PDB ID 2YP7; Gisaid no. EPI397688) (68–71). The sequence alignment is also annotated with additional lines to indicate residues comprising the receptor binding site (Receptor) (labeled r), the solvent-accessible surface residues (Srf res) (labeled s; determined by Areaimol, part of the CCP4 program suite [84, 85]), and the antigenic sites (labeled a, b, c, d, and e) (68–71).

FIG 3

Glycan binding to seal recHA. (A and B) Glycan array analysis of seal11 recHA (A) and A/harbor seal/New Hampshire/179629/2011 (B). Glycans on the microarray are grouped according to SA linkage: 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 asialo glycans (gray). The error bars reflect the standard error in the signal for six independent replicates on the array. The structures of the numbered glycans are found in Table 2. Specific glycan structures that were used in biosensor assays are represented on the array as glycans 19/20, 22, 52/53, and 56. (C) The binding kinetics of seal11 recHA protein to specific biotinylated glycans, 3-SLN-b, 3-SLNLN-b, 6-SLN-b, and 6-SLNLN-b, immobilized on streptavidin-coated biosensors were analyzed by BLI. The black vertical line indicates the switch in the experiment from collecting association to collecting dissociation data. (D) 3-SLN glycan interactions with seal11 HA RBS. Seal HA is shown in cartoon form, while 3-SLN and interacting HA residues are shown as sticks. Hydrogen bonds between the glycan and HA are shown as dashed lines. The structural figure was generated with MacPyMol (83).

FIG 4

Comparison of seal11 HA antigenic sites with other HA structures. (A) Recognized AS mapped onto the seal11 HA structure, shown as a surface representation and colored individually. (B) Structural comparison of the antigenic sites on the HA molecules between A(H3N8) (avian dkUkr63 and seal11) and seasonal H3N2 (Aichi68 and HK05). Three-dimensional models of the H3 HA molecules of each HA were used and are shown as surface representations. Expanded views of the antigenic sites (A through E) are shown. Amino acids are colored as follows: positive (Arg and Lys), blue; negative (Asp and Glu), red; hydrophobic (Ala, Phe, Gly, Ile, Leu, Met, Val, and Trp), yellow; polar (His, Asn, Gln, Ser, Thr, and Tyr), green.

FIG 5

Structure of seal NA. (A) Overall structure of one NA monomer, looking down on the monomer, with the position of the enzyme active site indicated. While only one glycosylation site was occupied in the final structure (pink sticks), two others that are visible in this view are labeled and shown as sticks. The fourth position, Asn84, was substituted in the protein but would be at the back of this view. (B) NA active site, with highly conserved residues shown as sticks (labeled in black text). The amino acid substitutions between seal11 NA (green) and the avian N8 NA (cyan) are labeled and shown as sticks. The seal residue is indicated first in the labels and the avian residue second. (C and D) Exploded views of the enzyme active site with NA antivirals Ose (C) and Zan (D) bound. The NA residues that interact with each drug (dashed lines) are shown as sticks.