H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes

Abstract

There are 15 subtypes of influenza A virus (H1-H15), all of which are found in avian species. Three caused pandemics in the last century: H1 in 1918 (and 1977), H2 in 1957 and H3 in 1968. In 1997, an H5 avian virus and in 1999 an H9 virus caused outbreaks of respiratory disease in Hong Kong. We have determined the three-dimensional structures of the haemagglutinins (HAs) from H5 avian and H9 swine viruses closely related to the viruses isolated from humans in Hong Kong. We have compared them with known structures of the H3 HA from the virus that caused the 1968 H3 pandemic and of the HA--esterase--fusion (HEF) glycoprotein from an influenza C virus. Structure and sequence comparisons suggest that HA subtypes may have originated by diversification of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection.

Fig. 1. Structure-based sequence alignment of influenza A H3, H5 and H9 HAs. Sequences of human H3hu (A/Aichi/2/68), avian H5av (A/Dk/Sing/97), human H5hu (A/HK/486/97), swine H9sw (A/Sw/9/98) and human H9hu (A/Hong Kong/1073/99). Residues identical to H3 are blank. Numbering is as in the human H3 sequence. Horizontal lines above the sequences indicate subdomains (red, F′ and F; yellow, E′; blue, R). Sequence differences between the H5 human and avian, and H9 swine and human viruses are boxed in red (H5) and blue (H9). An asterisk labels the arginine between HA1 and HA2 that is removed during proteolytic cleavage of HA0. The alignment was generated using GCG (Womble, 2000) and edited manually on the basis of the X-ray structures.

Fig. 2. H5 avian and H9 swine HA structures compared with H3 HA and HEF. (A) Ribbon diagram of the trimer of H5 (A/Dk/Sing/97) avian HA coloured by subdomains: receptor subdomain R (blue), vestigial enzyme subdomain E′ (yellow), HA2 stem F subdomain (red), F′ subdomain HA1 1–52 (pink), F′ subdomain HA1 275–307 (purple). Oligosaccharides are coloured by atom type. (B) Trimer of H9 swine (A/Sw/9/98) HA. (C) Monomer of H5 (A/Dk/Sing/97) avian HA. Helix A and B, the interhelical loop between them, and the fusion peptide in HA2 are labelled. N1 and C1 indicate the termini of HA1; N2 and C2, those of HA2. The trimer axis, receptor-binding site and two prominent antigenic loops, 140s and 150s, are labelled. Residue numbers mark oligosaccharides, which are coloured by atom type. Carbohydrate attachment sites are at HA1 21, 33, 169 and 289, and at HA2 154. (D) Monomer of the H9 (A/Sw/9/98) swine HA. The x marks the deleted 140s loop. Carbohydrate attachment sites are at HA1 21, 128, 210, 289 and 296, and at HA2 154. (E) Monomer of trimeric H3 (A/Aichi/2/68) human HA (Wilson et al., 1981). Carbohydrate attachment sites are at HA1 8, 22, 38, 81, 165 and 285, and at HA2 154. (F) Monomer of trimeric influenza C virus (C/Johannesburg/1/66) haemagglutinin–esterase–fusion (HEF) protein (Rosenthal et al., 1998; Zhang et al., 1999). These figures and Figures 3B, 4A, B and D were generated using MOLSCRIPT and BOBSCRIPT (Kraulis, 1991; Esnouf, 1997).

Fig. 3. Differences in subdomain locations in H3, H5 and H9 HAs and schematic diagram of key residues determining HA subtype structures. (A) R.m.s.ds between the α-carbon coordinates of the H5 and H3 HAs are plotted against HA sequence number. HA1 blue line = R subdomain superposition, yellow = E subdomain superposition, red = F′ subdomain superposition. An asterisk marks insertions in H5 relative to H3 (HA1 residues 53, 81, 95, 125, 133 and 255) and + marks a deletion at HA1 124. The underlines designate interface residues (F′–E′, orange; R–E′, green; F–E′, red; F–R, red) and the subdomain locations: E′, yellow; R, blue; and F and F′, red. (B) Superposition of the long (B) α-helices of HA2 showing differences in the intrahelical loop and long (B) α-helix N-terminal conformations: H9, blue; H5, red; H3, grey. Insert: schematic diagram indicating that the H5 and H9 globular domains are rotated 20° and displaced distally by 4 Å relative to the H3 HA. Within individual subdomains, the r.m.s.d. overlaps are 1.1, 1.0 and 1.1 Å for R, E′ and F′, respectively. The interhelical loop of HA2 contacts the R, E′ and F′ subdomains of HA1 (dashed line). (C) Sequence alignment of 15 HA subtypes at HA1 107 and HA2 60–90. Residues (*) suggested to determine the conformational differences between subtypes are shaded grey. The groups of subtypes (clades) predicted to have similar structures are shaded in four colours (grey, H3/H4/H14, G-75, K-88; yellow, H7/H10/H15, non-G-75, non-K-88, non-E-107; red, H1/H2/H5/H6/H11/H13, non-G-75, non-K-88, E-107; blue, H8/H9/H12, non-G-75, non-K-88, E-107, Q-87). The strains used to represent each subtype were: H1 (A/USSR/90/77), H2 (A/RI/5+/57), H3 (A/Aichi/2/68), H4 (A/Dk/Czechoslovakia/56), H5 (A/Dk/Singapore/3/97), H6 (A/Sh/Australia/1/72), H7 (A/Eq/Prague/1/56), H8 (A/Tk/Ontario/6118/68), H9 (A/Sw/HK/9/98), H10 (A/Ck/Germany/N/49), H11 (A/Dk/England/56), H12 (A/Dk/Alberta/60/76), H13 (A/Gl/Maryland/704/77), H14 (A/Ma/Gurjev/263/82) and H15 (A/Sh/West Australia/2576/79). (D) Phylogenetic relationships between HA subtypes and influenza B (B/Lee/40) HA based on HA amino acid sequences. The displacement of the HA1 subdomains (insert: HA viewed down the trimer axis from the top; the asterisk marks the interface where H3/H9 differ from H5 near residue 216) probably occurred when the H3, H4, H14 group diverged from the H7, H10 and H15 subtypes. The shaded colours for groups of subtypes correspond to the colours and groups in (C).

Fig. 4. H5 and H9 residues implicated in the low pH-induced refolding required for membrane fusion. (A) An H5 HA trimer with monomers coloured blue, pink and white. The box indicates approximately the area detailed in (B–D). Red segments of the fusion peptide and near HA1 Met30 are indicated by red dotted lines in (D). (B) H9 HA difference in electron density (contoured at 4σ) suggests an ion bound between three HA2 Lys106 residues at the centre of the triple-helical stem region in H9. HA2 Glu103 is hydrogen-bonded to both HA2 Lys51 and HA2 Lys106. A number of water molecules are found around the putative counter-ion and below HA2 Lys106 (not shown). (C) H5 HA2 His111 is buried by HA1 Leu320, HA2 Trp21 and Phe110, and is located near the fusion peptide (arrow), HA2 residues G1, L2, F3 coloured blue. (D) In the HA1–HA2 trimer, residue HA2 106 is accessible to solvent only through narrow channels (green arrows) after the fusion peptide (G1, L2 and F3) fits between the helices following HA0 cleavage to HA1 and HA2 (Chen et al., 1998). For clarity, one helix in the front of the cavity and peripheral residues were omitted. This figure was generated using GRASP (Nicholls et al., 1991).