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.
INTRODUCTION
Based on the serological reactivity of influenza A virus surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), 18 HA (H1 to H18) and 11 NA (N1 to N11) variants have been identified in aquatic birds and bats (1–3). The Centers for Disease Control and Prevention estimates that annually, seasonal influenza viruses can infect up to 20% of the human population (http://www.cdc.gov/flu). In the United States alone, estimates of influenza-associated deaths range from 3,000 to 49,000 per annum (4). In the last 100 years, three influenza virus subtypes have successfully adapted to the human population, causing four pandemics: H1N1 in 1918 and 2009, H2N2 in 1957, and H3N2 in 1968 (5–8). Avian influenza viruses have become endemic in domestic poultry in certain parts of the world, and a growing number of human cases of avian influenza infection with different subtypes [A(H5N1), A(H5N6), A(H7N2), A(H7N3), A(H7N7), and A(H9N2)] have been reported in recent years (9–14), causing significant public health concern.
During 2013, three novel avian influenza A viruses, A(H7N9), A(H10N8), and A(H6N1), were isolated from human patients. The first human infection with an avian influenza A(H7N9) virus was reported in March 2013 near Shanghai, China, and its emergence led to a human epidemic in that country (15). Indeed, as of October 2014, the virus had spread widely to a number of provinces and municipalities in China and resulted in over 450 human cases, with an ∼38% mortality rate. Not surprisingly, most research has focused on this A(H7N9) virus due to its rapid spread and severity. Whole-genome genetic analysis highlighted the fact that the six internal genes of the virus all originated from H9N2 viruses and may contribute to the increased pathogenicity observed in human infections (16).
In mid-2013, an influenza A(H6N1) virus was detected in a sample collected from a patient with flu-like symptoms in Taiwan (17). Although this A(H6N1) (A/Taiwan/2/2013) infection was the first reported human case, the A(H6N1) subtype avian influenza virus has been circulating throughout North America and Eurasia for many years (18, 19). Indeed, H6-specific antibodies have previously been detected in personnel working in Chinese live-animal markets as well as U.S. veterinarians exposed to birds (20, 21). Phylogenetic studies indicated that the A/Taiwan/2/2013 internal genes originated from different A(H6N1) Eurasian lineages, already circulating in chickens in Taiwan, rather than from the H9N2 internal genes seen in A(H7N9) viruses (16). A single Pro186Leu (in H3 numbering) substitution in the HA of the human isolate relative to the avian isolates was suggested to increase mammalian receptor binding (22). Indeed, a recent study (23) assessed the receptor-binding preference of a panel of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011 and found that ∼34% of the 257 viruses analyzed recognized the human type receptor using an established solid-phase binding assay (24).
In December 2013, China also reported to WHO the first of three human cases of disease caused by avian influenza A(H10N8) virus (25, 26), two of which were fatal. All three human cases were detected in Jiangxi Province, and evidence suggests a zoonotic source, as all three patients had visited live bird markets where the virus had been detected in poultry (27, 28). Although two human infections with A(H10N7) subtypes had been reported, with patients presenting with conjunctivitis or mild respiratory symptoms (29, 30), this was the first reported human infection with an influenza virus with an N8 subtype NA. Phylogenetic analysis of the virus isolated from the first fatal case (A/Jiangxi-Donghu/346/2013) revealed that all the genes of this virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses, as with H7N9 and H5N1 (25, 31, 32).
Fortunately, no sustained human-to-human transmission has been detected for these recent novel influenza subtypes, but the appearance of human infection with novel avian influenza (33) viruses has raised alarm within the public health community and has prompted a detailed assessment of each subtype for pandemic potential. In particular, characterization of the HA surface antigen is critical in that it binds to sialic acid (SA) oligosaccharides on the host epithelial cell surfaces and mediates virus uptake and subsequent infection. Whereas human seasonal influenza viruses bind to receptors containing α2-6-linked SA, avian influenza viruses predominantly bind to receptors containing α2-3-linked SA (34, 35). The recent A(H7N9) virus binds avian receptors but also has some propensity to bind to human receptors (36).
To better understand the structure and function of influenza virus H6 and H10 HAs, we recombinantly expressed the HA ectodomains from 4 viruses: an avian A(H6N2) virus, A/Chicken/New York/14677-13/1998 (avH6); a human A(H6N1) virus, A/Taiwan/2/2013 (huH6); an avian H10N7 virus, A/green-winged teal/Texas/Y171/2006 (avH10); and a human A(H10N8) virus, A/Jiangxi-Donghu/346/2013 (huH10). The receptor binding specificities of the HAs were analyzed by glycan microarray analysis. In addition, three-dimensional atomic structures were determined for each HA (apo form), including complex structures of avH6 HA in complex with an avian receptor analog (LS-tetrasaccharide a [LSTa]); huH6HA in complex with both an avian receptor analog (3′-sialyl-N-acetyllactosamine [3SLN]) and a human receptor analog (6′-sialyl-N-acetyllactosamine [6SLN]).
MATERIALS AND METHODS
Cloning.
The mature hemagglutinin (HA) ectodomains of avH6, huH6, avH10, and huH10 were codon optimized, synthesized, and cloned into the baculovirus transfer vector pAcGP67-B (BD Biosciences, San Jose, CA) in frame with an N-terminal baculovirus GP67 signal peptide and a C-terminal thrombin cleavage site, a “foldon” sequence (37), and a hexahistidine tag at the extreme C terminus of the fusion protein to enable purification (38).
Protein expression and purification.
Soluble HA was purified from culture supernatants by sequential metal affinity and gel filtration chromatography. Protein was used at this stage for glycan binding experiments. For structural analyses, proteins were prescreened for efficient cleavage of the foldon domain using thrombin and trypsin. As a result of these pilot studies, large-scale preparations of avH6 and avH10 were both subjected to thrombin digestion as described previously (39–41), while huH6 and huH10 were both subjected to trypsin digestion (1:1,000 [wt/wt] ratio of trypsin to protein). HA trimers were buffer exchanged into 10 mM Tris-HCl, 50 mM NaCl (pH 8.0), and concentrated to 15 to 18 mg/ml for crystallization trials.
Crystallization and data collection.
Initial crystallization trials were established using a Topaz free interface diffusion (FID) crystallizer system (Fluidigm Corporation, San Francisco, CA). Following optimization, diffraction quality crystals were obtained at 20°C using a modified microbatch under oil (42) with a reservoir solution containing 0.2 M ammonium fluoride and 20% (wt/vol) polyethylene glycol (PEG) 3350 for avH6, 0.01 M nickel chloride, 0.1 M Tris-HCl (pH 8.5), and 20% (wt/vol) PEG 2000MME for huH6, 0.1 M MES (morpholineethanesulfonic acid; pH 6.5) and 30% (vol/vol) PEG 300 for avH10, and 0.2 M potassium sodium tartrate and 20% (wt/vol) PEG 3350 for huH10. For H6 receptor analog complexes, crystals were soaked for 1 h in the crystallization buffer containing 10 mM LSTa, LSTc, 3SLN, or 6SLN (V-labs Inc., Covington, LA). However, attempts to soak the human analogs, 6SLN and LS-tetrasaccharide c (LSTc), dramatically decreased the crystal diffraction, and no diffraction-quality data could be collected. Crystals were flash-cooled at 100 K; data were collected at the Advanced Photon Source (APS) beamline 22ID or 22BM at 100 K and processed with the DENZO-SACLEPACK suite (43). Statistics for data collection are presented in Table 1.
TABLE 1.
Data collection and refinement statistics for the H6 and H10 HA crystal structures
| Characteristic | Result for virus ligand |
||||||
|---|---|---|---|---|---|---|---|
| chicken/New York/14677-13/1998 (avH6) |
Taiwan/2/2013 (huH6) |
A/green-winged teal/Texas/Y171/2006 (avH10) Apo | Jiangxi-Donghu/346/2013 (huH10) Apo | ||||
| Apo | LSTa | Apo | 3′ SLN | 6′ SLN | |||
| Data collection | |||||||
| Beamline collected | APS, 22-BM | APS, 22-ID | APS, 22-ID | APS, 22-ID | |||
| Space group | C2 | C2 | C2 | C2 | C2 | P212121 | C2 |
| Cell dimensions (Å) | 252.55, 134.82, 123.09 | 254.97, 134.78, 122.62 | 226.63, 100.61, 175.48 | 186.98, 100.08, 135.24 | 186.44, 99.58, 134.22 | 71.92, 144.63, 209.66 | 217.40, 217.61, 146.27 |
| Cell angle (°) | 90, 113.23, 90 | 90, 112.2, 90 | 90, 99.66, 90 | 90, 126.04, 90 | 90, 125.96, 90 | 90, 90, 90 | 90, 89.98, 90 |
| Resolution (Å) | 48.13–2.5 (2.589–2.5)a | 45.24–2.8 (2.90–2.8) | 50–2.4 (2.488–2.4) | 49.94–2.7 (2.796–2.7) | 49.77–3.022 (3.13–3.022) | 50–2.8 (2.9–2.8) | 50–2.7 (2.796–2.7) |
| Rsym (%) | 5.9 (20.2) | 8.3(24.6) | 9.4 (43.3) | 8.5 (29.1) | 12.7 (46.4) | 14.4 (70.2) | 8.4 (62.6) |
| I/σ | 30.6 (5.6) | 17.0 (4.7) | 17.7 (2.1) | 20.6 (4.0) | 13.1 (2.0) | 12.2 (2.8) | 18.2 (1.7) |
| Completeness (%) | 98.9 (94.3) | 98.5 (91.9) | 96.6 (81.3) | 98.3 (92.4) | 94.8 (84.0) | 98.8 (98.4) | 99.96 (100.00) |
| Redundancy | 3.8 (3.7) | 3.2 (2.8) | 3.7 (2.9) | 3.4 (3.3) | 3.2 (2.6) | 4.6 (4.7) | 3.9 (3.9) |
| Refinement | |||||||
| Resolution (Å) | 48.13–2.5 (2.59–2.50)a | 45.24–2.8 (2.90–2.80) | 50–2.4 (2.49–2.40) | 49.9–2.7 (2.80–2.7) | 49.8–3.02 (3.13–3.02) | 50–2.8 (2.9–2.8) | 50–2.7 (2.80–2.7) |
| No. of reflections (total) | 129,376 | 92,840 | 146,479 | 54,578 | 37,065 | 54,072 | 185,693 |
| No. of reflections (test) | 12,247 | 8,585 | 12,244 | 5,081 | 3,241 | 5,312 | 18,521 |
| Rwork/Rfree | 0.213/0.247 | 0.220/0.253 | 0.222/0.253 | 0.214/0.246 | 0.246/0.279 | 0.224/0.258 | 0.241/0.263 |
| No. of atoms | 24,430 | 23,442 | 24,510 | 12,006 | 11,923 | 11,676 | 46,260 |
| RMSD | |||||||
| Bond length (Å) | 0.014 | 0.013 | 0.015 | 0.014 | 0.006 | 0.012 | 0.01 |
| Bond angle (°) | 1.47 | 1.57 | 1.66 | 1.79 | 1.23 | 1.6 | 1.43 |
| MolProbityb scores (%) | |||||||
| Favored | 97 | 95 | 96 | 95 | 93 | 95 | 94 |
| Outliers | 0.28 | 0.38 | 0.51 | 0.95 | 0.82 | 0.34 | 0.67 |
| PDB code | 4WSR | 4WSS | 4WST | 4WSU | 4WSV | 4WSW | 4WSX |
Numbers in parentheses refer to the highest-resolution shell.
From reference 42.
Structure determination and refinement.
All structures were determined by molecular replacement with Phaser (44). For avH6, the H2 structure from A/Japan/305/1957 (PDB code 2WRD) (HA0, 61% identity) was used as the search model, while the H7 structure from A/New York/107/2003 (PDB code 3M5G) (HA1, 52% identity; HA2, 80% identity) was used for avH10. For huH6 and huH10, structures were determined by molecular replacement using the avH6 and avH10 as the search models. Solution models were “mutated” to the correct sequence, rebuilt by Coot (45), and refined with PHENIX (46) and REFMAC using translation/libration/screw (TLS) refinement (47). The final structural models were assessed using MolProbity (48), and their refinement statistics are presented in Table 1.
Glycan binding analyses.
Glycan microarray printing and recombinant hemagglutinin (recHA) analyses have been described previously (39). Briefly, HA-antibody precomplexes were prepared by mixing HA (10 μl, 1 mg/ml), mouse anti-penta-His-Alexa Fluor 488 (17.5 μl, 0.2 μg/ml; Qiagen Inc.), and anti-mouse-IgG-Alexa Fluor 488 (1.2 μl, 2 mg/ml; Life Technologies) in a molar ratio of 4:2:1, respectively. Mixtures were incubated for 60 min on ice, then diluted with 500 μl phosphate-buffered saline (PBS) buffer containing 2% (wt/vol) bovine serum albumin and streptavidin-Alexa Fluor 488 (1:1,000 [vol/vol]; Life Technologies), and incubated on the microarray slide on ice for 60 min. Slides were subsequently washed by successive rinses in PBS with 0.05% Tween 20 (vol/vol), PBS, and deionized water and then dried. Fluorescence intensities were detected using a ProScanArray HT microarray scanner (Perkin-Elmer), and subsequent image analyses were carried out with ImaGene 9 software (BioDiscovery). Table 2 presents the specific glycans on the arrays.
TABLE 2.
Glycan microarray for human and avian H6 and H10 HAs
| Category and glycan | Structure | Bindinga |
|||
|---|---|---|---|---|---|
| huH6 | avH6 | huH10 | avH10 | ||
| Sialic acid | |||||
| 1 | Neu5Acα | NB | NB | NB | NB |
| 2 | Neu5Acα | NB | NB | NB | NB |
| 3 | Neu5Acβ | NB | NB | NB | NB |
| α2-3 sialosides | |||||
| 4 | Neu5Acα2-3[6OSO3]Galβ1-4GlcNAcβ | NB | NB | + | + |
| 5 | Neu5Acα2-3Galβ1-3[6OSO3]GalNAcα | NB | + | +++ | +++ |
| 6 | Neu5Acα2-3Galβ1-4[6OSO3]GlcNAcβ | NB | NB | +++ | +++ |
| 7 | Neu5Acα2-3Galβ1-4(Fucα1-3)[6OSO3]GlcNAcβ-propyl-NH2 | NB | + | + | NB |
| 8 | Neu5Acα2-3Galβ1-3[6OSO3]GlcNAc-propyl-NH2 | NB | NB | +++ | +++ |
| 9 | Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4)GlcNAcβ | +++ | +++ | +++ | +++ |
| 10 | Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-6)GalNAcα | NB | +++ | +++ | +++ |
| 11 | Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ | +++ | +++ | +++ | +++ |
| 12 | Neu5Acα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[Neu5Acα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ | +++ | +++ | +++ | +++ |
| 13 | Neu5Acα2-3Galβ | NB | + | + | NB |
| 14 | Neu5Acα2-3Galβ1-3GalNAcα | NB | +++ | +++ | +++ |
| 15 | Neu5Acα2-3Galβ1-3GlcNAcβ | NB | + | +++ | +++ |
| 16 | Neu5Acα2-3Galβ1-3GlcNAcβ | NB | +++ | +++ | +++ |
| 17 | Neu5Acα2-3Galβ1-4Glcβ | NB | + | + | + |
| 18 | Neu5Acα2-3Galβ1-4Glcβ | NB | +++ | + | + |
| 19 | Neu5Acα2-3Galβ1-4GlcNAcβ | NB | +++ | + | +++ |
| 20 | Neu5Acα2-3Galβ1-4GlcNAcβ | + | +++ | + | + |
| 21 | Neu5Acα2-3GalNAcβ1-4GlcNAcβ | NB | + | NB | NB |
| 22 | Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ | +++ | +++ | +++ | +++ |
| 23 | Neu5Aca2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ | NB | +++ | +++ | +++ |
| 24 | Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ | + | +++ | +++ | +++ |
| 25 | Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ | + | +++ | +++ | +++ |
| 26 | Neu5Acα2-3Galβ1-3GalNAcα | NB | NB | +++ | +++ |
| 27 | Galβ1-3(Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-6)GalNAcα | NB | NB | NB | NB |
| 28 | Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ | NB | +++ | NB | NB |
| 29 | Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ | NB | +++ | NB | NB |
| 30 | Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ | + | +++ | NB | NB |
| 31 | Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ | NB | +++ | + | + |
| 32 | Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ | NB | +++ | NB | NB |
| 33 | Neu5Acα2-3Galβ1-3[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ | + | +++ | + | + |
| 34 | Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ | +++ | +++ | + | +++ |
| 35 | Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 36 | Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 37 | Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ | NB | NB | NB | NB |
| 38 | Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ | NB | NB | NB | NB |
| 39 | Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ | NB | NB | NB | NB |
| 40 | Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ | NB | NB | NB | NB |
| α2-6 sialosides | |||||
| 41 | Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ | NB | NB | NB | NB |
| 42 | Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 43 | Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 44 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 45 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-6]-Manβ1-4GlcNAcβ1-4GlcNAcβ | + | NB | NB | NB |
| 46 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]GalNAca | NB | NB | NB | NB |
| 47 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3[Neu5Acα2-6Galβ1-4GlcNAcβ1-6]GalNAca | NB | NB | NB | NB |
| 48 | Neu5Acα2-6GalNAcα | NB | NB | NB | NB |
| 49 | Neu5Acα2-6Galβ | NB | NB | NB | NB |
| 50 | Neu5Acα2-6Galβ1-4Glcβ | NB | NB | NB | NB |
| 51 | Neu5Acα2-6Galβ1-4Glcβ | NB | NB | NB | NB |
| 52 | Neu5Acα2-6Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 53 | Neu5Acα2-6Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 54 | Neu5Acα2-6GalNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 55 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3GalNAcα | NB | NB | NB | NB |
| 56 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 57 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3GalNAcα | NB | NB | NB | NB |
| 58 | Neu5Aca2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 59 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ | NB | NB | NB | NB |
| 60 | Galβ1-3(Neu5Acα2-6)GlcNAcβ1-4Galβ1-4Glcβ-Sp10 | NB | NB | NB | NB |
| 61 | Neu5Acα2-6[Galβ1-3]GalNAca | NB | NB | NB | NB |
| 62 | Neu5Acα2-6Galβ1-4GlcNAcβ1-6[Galβ1-3]GalNAca | NB | NB | NB | NB |
| 63 | Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6[Galβ1-3]GalNAca | NB | NB | NB | NB |
| Mixed α2-3 and α2-6 biantennaries | |||||
| 64 | Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 65 | Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | + | +++ | + |
| 66 | Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα | NB | + | +++ | +++ |
| 67 | Neu5Acα2-3(Neu5Acα2-6)GalNAcα | NB | NB | NB | NB |
| N-Glycolylneuraminic acid glycans | |||||
| 68 | Neu5Gcα | NB | NB | NB | NB |
| 69 | Neu5Gcα2-3Galβ1-3(Fucα1-4)GlcNAcβ | NB | NB | NB | NB |
| 70 | Neu5Gcα2-3Galβ1-3GlcNAcβ | NB | NB | NB | NB |
| 71 | Neu5Gcα2-3Galβ1-4(Fucα1-3)GlcNAcβ | NB | + | NB | NB |
| 72 | Neu5Gcα2-3Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 73 | Neu5Gcα2-6GalNAcα | NB | NB | NB | NB |
| 74 | Neu5Gcα2-6Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| α2-8-linked sialosides | |||||
| 75 | Neu5Acα2-8Neu5Acα | NB | NB | NB | NB |
| 76 | Neu5Acα2-8Neu5Acα2-8Neu5Acα | NB | NB | NB | NB |
| 77 | Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ | NB | NB | NB | NB |
| 78 | Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ | NB | NB | NB | NB |
| 79 | Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ | NB | NB | NB | NB |
| 80 | Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ | NB | NB | NB | NB |
| 81 | Neu5Acα2-8Neu5Acβ-Sp17 | NB | NB | NB | NB |
| 82 | Neu5Acα2-8Neu5Acα2-8Neu5Acβ | NB | NB | NB | NB |
| β2-6-linked and 9-O-acetylated sialic acids | |||||
| 83 | Neu5Acβ2-6GalNAcα | NB | NB | NB | NB |
| 84 | Neu5Acβ2-6Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 85 | Neu5Gcβ2-6Galβ1-4GlcNAc | NB | NB | NB | NB |
| 86 | Galβ1-3(Neu5Acβ2-6)GalNAcα | NB | NB | NB | NB |
| 87 | [9NAc]Neu5Acα | NB | NB | NB | NB |
| 88 | [9NAc]Neu5Acα2-6Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| Asialo glycans | |||||
| 89 | Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 90 | Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ | NB | NB | NB | NB |
| 91 | Galβ1-4GlcNAcβ1-2Manα1-3[Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAcβ | NB | NB | NB | NB |
| 92 | GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ | NB | NB | NB | NB |
| 93 | GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ | NB | NB | NB | NB |
| 94 | Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ | NB | NB | NB | NB |
| 95 | Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ | NB | NB | NB | NB |
| 96 | Galβ1-3GalNAcα | NB | NB | NB | NB |
Significant binding of samples to each glycan was qualitatively estimated based on the relative strength of the signal for the data. Symbols: +, relative fluorescence intensity of 2,000 to 4,999; ++, relative fluorescence intensity of 5,000 to 9,999; +++, relative fluorescence intensity of >10,000; NB, no binding or relative fluorescence intensity of <1,999.
For kinetic studies, biotinylated receptor analogs, Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcβ-biotin (3SLNLN-b) and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcβ-biotin (6SLNLN-b), were obtained from the Consortium for Functional Glycomics (www.functionalglycomics.org) through the resource request program. Glycans were precoupled to streptavidin-coated biosensors (Fortebio Inc.), and binding of recombinant HA, diluted to 0.23 μM trimer in kinetics buffer (PBS containing 0.02% Tween 20 and 100 μg/ml bovine serum albumin), was analyzed by bio-layer interferometry (BLI) using an Octet Red instrument (Fortebio, Inc.) according to the manufacturer's instructions. Data were analyzed using the system software and fitted to a 1:1 binding model.
PDB accession codes.
The atomic coordinates and structure factors of each of the seven HA models described in this study are available from the Research Collaboratory for Structural Bioinformatics (RCSB) PDB under accession codes 4WSR to 4WSX (Table 1).
RESULTS AND DISCUSSION
Overall structure of H6 and H10 HAs.
The three-dimensional HA structures of the trimeric ectodomain from avH6, huH6, avH10, and huH10 HAs were determined by X-ray crystallography (Table 1). The HA protein is synthesized as a single-chain precursor (HA0) during viral replication and then cleaved by a specific host protease into the infectious HA1 and HA2 forms. In the baculovirus expression system used for these studies, all HAs were produced in the HA0 form. For structural studies, both huH6 and huH10 HA proteins were digested with trypsin to remove the trimerization tag, which also cuts the HAs into the active HA1/HA2 form. Both avH6 and avH10 HAs were digested with thrombin to remove the trimerization tag but retained the HA0 form. However, while the avH6 has an intact cleavage site, visualized as a loop in the final structure, the avH10 loop was disordered and could not be built in the final model.
As expected, the overall structure of the HA monomer is composed of a globular head containing the receptor binding site (RBS) and a membrane-proximal domain that includes a central helical stalk and the HA1/HA2 cleavage site (39–41, 49–56). For avH6 HA, five asparagine-linked glycosylation sites (NXS/T) are predicted in the HA monomer (Asn residues 11, 23, 290, 295, and 482 in avH6 numbering) (Fig. 1), while one extra site is predicted for huH6 HA at Asn167 (huH6 numbering) (Fig. 1) in the HA1. Interpretable carbohydrate electron density, containing only one or two N-acetylglucosamines, was observed at Asn23 and Asn290 in the HA1 for avH6 HA and at Asn11, Asn23, and Asn167 in the HA1 and at Asn154 in the HA2 for huH6 HA (Fig. 2A). Both avH10 and huH10 have the same six predicted glycosylation sites with interpretable carbohydrate electron density, observed at Asn12, Asn28, and Asn235 in the HA1 and Asn82 and Asn154 in the HA2 for avH10 and at Asn235 in the HA1 and Asn82 in the HA2 for huH10 (Fig. 2B).
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).
Each of these virus HAs originates from distinct lineages that are geographically distinct, and thus significant sequence differences exist (83% between avH6 and huH6; 92% between avH10 and huH10). However, despite these sequence differences, a comparison of avH6 HA with huH6 HA and avH10 HA with huH10 HA revealed high similarity, with the Cα atoms superimposing, to give root mean square deviations (RMSD) of 0.66 Å and 0.75 Å, respectively (Table 3). When compared to H10 HA structures recently published by Vachieri et al. (57), both our avH10 and huH10 HAs revealed high similarities with their counterparts, with the Cα atoms superimposing, to give RMSD of 0.63 Å and 0.57 Å, respectively. Comparison of the H6 and H10 HA monomers to all human isolate H1 HA (PDB code 4LXV) (58), H3 HA (PDB code 2YP2) (59), H5 HA (PDB code 2FK0) (54), and H7 HA (PDB code 4LN6) (38) also revealed highly similar structures (Table 3). Based on their molecular phylogenies, HAs are divided into two groups and six clades: group 1 includes H1, H2, H5, and H6; H8, H9, and H12; H11, H13, and H16; and H17 and H18; group 2 includes H3, H4, and H14 and H7, H10, and H15 (53, 60). In agreement with this grouping, RMSD analyses revealed the H6 and H10 structures to be more structurally related to other HAs from the same group.
TABLE 3.
Comparison of RMSD for HA monomers
| Structurea | RMSD (Å) |
|||
|---|---|---|---|---|
| avH6 | huH6 | avH10 | huH10 | |
| 4LXV_H1 | 1.2 | 1.34 | 1.68 | 1.69 |
| 2YP2_H3 | 1.81 | 1.65 | 1.29 | 1.29 |
| 2FK0_H5 | 1.25 | 1.36 | 1.51 | 1.64 |
| 4LN6_H7 | 1.84 | 1.61 | 0.88 | 0.71 |
| avH6 | ||||
| huH6 | 0.66 | |||
| avH10 | 1.56 | 1.43 | ||
| huH10 | 1.71 | 1.54 | 0.75 | |
For published structures used in the analysis, PDB codes are given.
Structural and functional analyses of RBSs.
The receptor binding site (RBS) is at the membrane-distal end of each HA monomer, and its specificity for sialic acid and the nature of its linkage to a vicinal galactose residue are major determinants of host range restriction (61). The consensus RBS for all current HAs is composed of three major structural elements: a 190 helix (residues 188 to 194 based on H3 numbering), a 220 loop (residues 221 to 228), and a 130 loop (residues 134 to 138). Highly conserved residues (Tyr98, Trp153, His183, and Tyr195) are also present in the base of the pocket (Fig. 2C). Similar to H7 HAs, H10 HAs also have an elongated 150 loop that also stretches into the receptor binding site (Fig. 2C) (38).
Previously, mutations in the HA receptor binding domains of A(H1N1) (Glu190Asp/Gly225Asp) and A(H2N2)/A(H3N2) (Gln226Leu/Gly228Ser) subtypes contributed to the human adaptation of these viruses to the 1918, 1957, and 1968 pandemic strains (55, 62–64). HAs from avH6, avH10, and huH10 maintain avian receptor binding motifs at the equivalent sites for these residue 190/225 and 226/228 constellations (Fig. 1). Interestingly, previous receptor binding studies of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011, using the solid-phase binding assay, revealed that ∼34% of the viruses analyzed in the study had the ability to recognize human type receptors (25). In addition, a recent publication on H10 HAs suggested that H10 virus had a high avidity for human receptors (57). To assess these observations and gain further insight into the interactions of H6 and H10 viruses with host receptors, glycan-binding analyses of avH6, huH6, avH10, and huH10 recHAs were performed. Glycan microarray analysis of all recHAs revealed a strong binding preference for the α2-3-linked sialosides (Table 2 and Fig. 3A to D). The avH6, huH6, and huH10 recHAs (Fig. 3A, B, and C) had limited binding to mixed α2-3/α2-6 branched sialosides (glycans 65 to 66), while the huH6 recHA had weak binding to one branched α2-6-linked glycan with a tri-LacNAc repeat (number 45) (Table 2 and Fig. 3B). Interestingly, huH6 also had significantly more reduced and restricted binding to the α2-3-linked sialosides on the array than avH6. Strong binding signals were detectable only for branched and longer sialosides (glycans 9, 11, 12, and 34). The huH6 HA also exhibited binding discrimination between the linkage present between position 2 (Gal) and 3 (GlcNAc and GalNAc) of the terminal trisaccharide, preferring a β1-4 linkage over a β1-3 linkage.
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.
Glycan binding to these recHAs was further analyzed by bio-layer interferometry (BLI) using an Octet Red system (Fortebio Inc.). The results of the binding of the H10 recHAs to biotinylated glycans (3SLNLN-b and 6SLNLN-b) preloaded onto streptavidin-coated biosensors confirmed the glycan array data in that both the avH10 and huH10 recHAs bound only to the α2-3-linked 3SLNLN-b analog, with apparent KDs of approximately 22.4 and 26.5 μM for the avian and human H10 HAs, respectively (Fig. 3E and Table 4). While no glycan binding was observed for huH6 recHA in this assay, the avH6 bound strongly to the 3SLNLN analog (Fig. 3E) and weakly to the human 6SLNLN analog (Fig. 3F).
TABLE 4.
Kinetic results for glycan binding to H6 and H10 recHAsa
| HA | Glycan | Apparent KD (μM) | kon (ms−1) | kobs (10−2 s−1)b | koff (10−3 s−1)b |
|---|---|---|---|---|---|
| AvH6 | 3SLNLN-b | 0.36 | 21,780 | 4.11 ± 0.11 | 9.79 ± 0.071 |
| 6SLNLN-b | 1.27 | 12,320 | 2.98 ± 0.05 | 15.6 ± 0.376 | |
| huH6 | 3SLNLN-b | ND | ND | ND | ND |
| 6SLNLN-b | ND | ND | ND | ND | |
| AvH10 | 3SLNLN-b | 22.41 | 930 | 2.18 ± 0.11 | 20.9 ± 0.113 |
| 6SLNLN-b | ND | ND | ND | ND | |
| huH10 | 3SLNLN-b | 26.52 | 570 | 1.56 ± 0.06 | 14.9 ± 0.828 |
| 6SLNLN-b | ND | ND | ND | ND |
Abbreviations: KD, dissociation constant; kon, association rate; kobs, observed rate; koff, dissociation rate; ND, not determined due to low binding and/or no binding.
Values are means ± standard errors.
To understand from a structural perspective how H6 HAs interact with host receptors, we solved the structures of avH6 HA in complex with an avian analog, LSTa, and huH6 HA in complex with both the avian 3SLN analog (Fig. 4C) and the human 6SLN analog (Fig. 4D). For avH6 HA with LSTa, the electron density maps revealed well-ordered features for only three of the five monosaccharides, SA-1, Gal-2, and GlcNAc-3, in the complex structure. Hydrogen bonds are formed between SA-1 and the residues Tyr91, Val131, Asn133, and His180 within the pocket (Fig. 4B). For huH6 HA with 3SLN, the electron density maps revealed well-ordered features for all components, SA-1, Gal-2, and GlcNAc-3, in the complex structure. Hydrogen bonds were formed between SA-1 and residues including Tyr91, Val131, Thr132, Asn133, Gln224, and Ser226 within the pocket (Fig. 4C). For huH6 HA with 6SLN, and in accordance with the poor binding to human glycans observed in our glycan binding studies, there was interpretable electron density only for SA-1 in the binding pocket. The SA-1 location is almost identical to that of SA-1 in the 3SLN complex structure, and hydrogen bonds were formed between SA-1 and residues equivalent to those in the 3SLN complex structure (Fig. 4A).
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.
Our data suggest that both the H6 and H10 recHAs have very limited specificity for human receptors and would require additional mutations to switch, such as those that occurred in previous human pandemic viruses. Vachieri et al. (57) noted that the switch from avian to human receptors for pandemic viruses was achieved by decreasing avidity for the avian receptor. While our studies did not show an overall decrease in avian receptor avidity between the two H10 HAs, the huH6 HA did have a significant drop in signal intensities for α2-3-linked sialosides compared to the avH6 HA, and differential binding was also observed for glycan linkage preference. In addition, our BLI studies revealed that the avH6 virus possesses weak binding to the human 6SLNLN analog. This may suggest that these A(H6N1) viruses possess an RBS that could switch to a human receptor preference with minimal changes. Interestingly, a recent study also found that ∼34% of the 257 H6 viruses isolated in southern China between 2008 and 2011 recognized the human type receptor in a solid-phase binding assay (23).
Indeed, the region of the huH6 HA RBS has some interesting features. Compared to avH6 HA, the huH6 HA has a single proline amino acid insertion at 136 (140 in H3 numbering), and as a result, a slightly longer loop is observed at this position. However, it is sufficiently far from the RBS that it may not affect receptor binding (Fig. 4A). HuH6 also has a further two substitutions, Pro184Leu (186 in H3 numbering) and Glu188Val (190 in H3 numbering) near the RBS (Fig. 4A). The stronger hydrophobicity of leucine and valine has been proposed to increase the hydrophobicity of the RBS, making the HA more likely to bind the human receptor (65). The huH6 HA also has a Ser226 (228 in H3 numbering) (Fig. 4A), which may constitute an intermediate state of the Gln224Leu-Gly226Ser constellation (226 and 228 in H3 numbering) which was observed in host receptor switching for both the H2N2 and H3N2 pandemic viruses. Interestingly, H6 viruses with this 226 change are endemic and have been the predominant A(H6N1) virus clade in chickens in Taiwan since 2005 (33). A Ser226 was also described in a swine A(H6N6) virus from China (25). It remains to be seen how effective an additional Gln224Leu mutation would be to affect a switch to human receptor binding. However, it is a position that should be closely monitored in ongoing and future H6 virus surveillance activities.
Conclusion.
Our detailed molecular characterization of recombinant HAs of H6 and H10 from both avian and human origins suggests that the HAs from influenza H6 and H10 virus human infections continue to maintain a strong avian receptor binding preference, thus reducing their potential to infect humans. However, the novel subtypes of avian influenza viruses exposed to the human population continue to be a major public health concern. Therefore, continued surveillance is needed as part of ongoing pandemic preparedness activities.
ACKNOWLEDGMENTS
This study was funded by the Centers for Disease Control and Prevention. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract DE-AC02-06CH11357. Glycan microarrays were produced for the Centers for Disease Control by the Consortium for Functional Glycomics (CFG), funded by National Institute of General Medical Sciences grant GM62116.
We thank the staff of SER-CAT sector 22 at APS for their help with data collection.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.
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