Unique Structural Features of Influenza Virus H15 Hemagglutinin

Abstract

Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.IMPORTANCE In the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.

Keywords: H15 subtype; X-ray crystallography; glycan arrays; influenza virus; receptor binding.

FIG 1

The amino acid sequence of the elongated 260-loop and the receptor binding specificity of the H15N9 shWA79 HA. (A) Schematic representation of the phylogenetic tree of influenza A virus HAs. The HAs are divided into group 1 and group 2, each of which can be further subdivided into subgroups. (B) Alignment of the HA 260-loop sequences of representative influenza viruses from all influenza A virus HA subtypes. The alignment indicates a marked elongation of the 260-loop of H15 HA compared to other HAs. The sequences of the group 1 and group 2 HAs are colored green and purple, respectively. (C) Glycan microarray analysis of the receptor binding specificity of recombinant H15N9 shWA79 HA indicates avian-type specificity and preferential binding of short, sulfated linear glycans (glycan numbers 11 to 15) and long, branched, O-linked and N-linked glycans. The A/Viet Nam/1203/2004 H5N1 and the A/Wyoming/3/2003 H3N2 HAs were used as controls for α2-3- and α2-6-linked sialoside binding, respectively. The mean signal (in relative fluorescence units [RFU]) and standard error were calculated from six independent replicates on the array. α2-3-linked sialosides are shown by yellow bars (glycans 11 to 79 on the x axis), and α2-6-linked sialosides are shown by green bars (glycans 80 to 135). Glycans 1 to 10 are nonsialylated control glycans and shown in gray. Glycans imprinted on the array are listed in Table S1 in the supplemental material.

FIG 2

Crystal structure of the H15N9 shWA79 HA. (A) Schematic cartoon representation of the H15N9 shWA79 HA trimer. One protomer is colored in blue and magenta for the HA1 and the HA2 subunits. The RBS and the 260-loop are marked with red rectangles. N-linked glycans that could be modeled in the electron density maps and the corresponding asparagine are shown as sticks and labeled in black. (B) Schematic representation of the RBS of the H15 HA. The key residues for receptor binding are shown as sticks and colored in blue. (C) The wider head domain of the H15 HA trimer is shown in the top and side view. Surface representations of the group 2 H15, H7, H3, H10, and H14 HA trimers indicate that the H15 head domain subunits tilt more outwards relative to the stem region compared to other subtypes.

FIG 3

The elongated 150- and 260-loops of the H15 HA. (A) The conformation of the 150-loop of the group 2 HAs. Superposition of the RBS subdomains of the H15N9 shWA79 HA (blue), A/Jiangxi/IPB13/2013 H10N8 HA (pink) (PDB entry 4XQ5), A/Shanghai/02/2013 H7N9 HA (orange) (PDB entry 4N5J), and pandemic H3N2 HA (gray) (PDB entry 4NFK). The conserved structural elements of the HA RBS (130-loop, 150-loop, 190-helix, and 220-loop) are labeled, and the side chains of H15 Asn158a, H10 Lys158a and H7 Asp158a are shown as sticks. (B) A model of the complex between H15 HA (blue and purple) and F045-092 Fab that targets the RBS (gray) indicates potential steric clashes between the extended 150-loop (black) and the variable region of the F045-092 Fab. The model was constructed by superposition of H15 HA and A/Victoria/3/1975 (H3N2) HA1 in complex with Fab045-092 (PDB entry 4O58) (30). (C) Conformation of the extended 260-loop of H15N9 shWA79 HAs. Surface representation of one HA protomer of the H15N9 shWA79 shows the extended 260-loop of H15 HAs (left). The HA protomer of the A/Shanghai/02/2013 H7N9 HA (right) is shown for comparison. The HA1 and HA2 subunits are colored blue and magenta, respectively, and the 260-loop is shown in red. (D) The elongated 260-loop of shWA79 H15N9 extends toward the base of the head domain and creates polar interactions with the 80-loop, 120-loop, and 180-loop. The HA head domain is colored blue, the stem domain is magenta, and the elongated 260-loop is red. The residues that appear to stabilize the conformation of the 260-loop are shown as sticks.

FIG 4

Models of complexes between H15 HA and CR8020, CR9114, H5M9, and 2D1 Fabs (gray) indicate that the elongated 260-loop (red) of the H15 HA can affect binding of antibodies to the lower part of the HA head (H5M9), but not to the HA stem (CR8020 and CR9114) or upper part of the HA head (2D1). For stem binding antibodies, the H15 HA2 chain was superimposed onto the HA2 chain of A/Hong_Kong/1/1968 (H3N2) (for CR8020, PDB entry 3SDY) (31) and A/Viet Nam/1203/2004 (H5N1) (for CR9114, PDB entry 4FQI) (32). For head binding antibodies, the H15 HA1 chain was superimposed onto the HA1 chain of A/Viet Nam/1203/2004 (H5N1) (for H5MP, PDB entry 4MHH) (34) and A/Brevig Mission/1/1918 (H1N1) (for 2D1, PDB entry 3LZF) (33).

FIG 5

Crystal structure of the H15N9 shWA79 HA in complex with an avian receptor analog. (A) Electron density maps (2Fo − Fc; top) and unbiased 2Fo-Fc electron density map (bottom) of avian receptor analog 3′-SLN in the crystal structure of the H15N9 shWA79 HA contoured at a 1σ level. The glycan structure of the 3′-SLN analog is represented above. Abbreviations: Sia, sialic acid; Gal, galactose; GlcNAc, N-acetylglucosamine. (B) Representation of the interactions between 3′-SLN and the H15 HA RBS. The RBS conserved structural elements are labeled and shown as cartoons. Selected residues and the receptor analog are labeled and shown as sticks. (C) The avian analog 3′-SLN in crystal structures with H10 and H15 HAs adopts a similar trans conformation, but in the H7 3′-SLN complex, it binds in a cis conformation (with regard to the C-1_Sia–C-2_Sia–O–C-3_Gal bond). The H7 HA contains Leu at position 226, while the H10 and H15 HAs have Gln. The superposition was performed on the sialic acid of the sialoside.

FIG 6

Glycan microarray assay of the receptor binding specificities of the H15 HA RBS mutants. The receptor binding specificity of recombinant HAs of the H15N9_E190D-G225D (A) and the H15N9_Q226L-G228S (B) double mutants were examined using the custom sialoside microarray. Mutations in the RBS of the H15 HA that are associated with a switch in receptor binding specificity of pandemic H1/H2/H3 viruses did not switch receptor binding specificity of the H15 HA. The mean signal and standard error were calculated from six independent replicates on the array. α2-3-Linked sialosides are shown as yellow bars (glycans 11 to 79 on the x axis), and α2-6-linked sialosides are shown as green bars (glycans 80 to 135). Glycans 1 to 10 are nonsialylated control glycans and shown in gray. Glycans imprinted on the array are listed in Table S1.