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J Virol. 2012 August; 86(16): 8645–8652.
PMCID: PMC3421765

Structure and Receptor Complexes of the Hemagglutinin from a Highly Pathogenic H7N7 Influenza Virus


Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in the Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutinin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild-type recombinant NL219 HA to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. The results suggest that the additional glycosylation sequon increases binding affinity to avian-type α2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens, which calls attention to the need for further studies to fully understand the unique properties of these viruses.


Influenza is a serious global public health concern. Each year up to 20% of the human population is infected with circulating influenza A virus, and in the United States estimates of influenza-associated deaths ranging from 3,000 to 49,000 per annum have been reported (48). Although there are three types of influenza virus (A, B, and C), only type A accounts for all known recent pandemics and most severe epidemics. Influenza A viruses are classified into subtypes according to the serological reactivity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (53). To date, 16 HAs (H1 to H16) and 9 NAs (N1 to N9) have been identified (16), and while all of these subtypes can be found in wild aquatic birds, only three subtypes in the last 100 years have adapted to the human population to cause four pandemics: H1N1 in 1918 and most recently 2009, H2N2 in 1957, and H3N2 in 1968 (20, 24, 42). Some subtypes, e.g., H5N1, H6N1, H7N2, and H9N2, have become endemic in domestic poultry in certain parts of the world (5).

Recent outbreaks in poultry involving viruses from some subtypes (H5, H7, and H9) have resulted in human infections, but their low transmissibility among humans has thus far prevented any new epidemics (11, 36, 47). These outbreaks, however, are a major concern for public health, particularly for H5N1 viruses, which has spread through wild and domestic bird populations across Asia, into Europe, the Middle East, and into Africa, causing hundreds of human infections with high fatality proportions. However, the threat of subtype H7 influenza viruses should not be underestimated. Cases of human infection by H7 influenza viruses have been reported sporadically since 1979 (51), caused by both low- and high-pathogenicity avian influenza (LPAI and HPAI, respectively) H7 viruses of the Eurasian and North American lineages. Outbreaks associated with human infections were reported in 2002 and 2003 in the United States (7, 8), 2004 in Canada (7, 23, 49), in 1995 and 2006-2007 in the United Kingdom (1, 15, 27, 33), in 2002 in Italy (37), and in 2003 in the Netherlands (17, 25). The 2003 outbreak in the Netherlands was caused by an HPAI H7N7 subtype and was the source of infection for 89 people exposed to affected poultry, including three cases of possible human-to-human virus transmission (17). Although most cases presented with conjunctivitis and/or mild influenza-like illness, one patient developed severe pneumonia leading to acute respiratory distress syndrome and death (17). The virus isolated from this fatal human case, A/Netherlands/219/2003 (NL219), is the subject of the present study.

When the virus from the fatal case was compared to isolates from nonfatal human infections from the same outbreak, A/Netherlands/33/2003 (NL33), 15 amino acid substitutions were identified, distributed among the basic polymerase 2 (PB2), acidic polymerase (PA), HA, NA, and nonstructural protein 1 (NS1) (17). The Glu627Lys substitution in the PB2 of NL219 virus was reported previously as a main determinant of virus pathogenicity and tissue distribution in a mouse model, while a Ala125Thr substitution on the HA generated a consensus motif for N-linked glycosylation of Asn123. This change was correlated with increased replication efficiency and wider tissue distribution of NL219 virus (HA numbering used herein is based on the mature protein) (13, 32).

The HA glycoprotein binds to oligosaccharide host cell surface receptors containing sialic acid (SA) and subsequently mediates virus uptake and membrane fusion. 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 (29, 38). In an attempt to better understand the molecular characteristics of the NL219 HA, we determined its three-dimensional atomic structure and that of its complexes with an avian receptor analog (3′-sialyl-N-acetyllactosamine [3′SLN]) and a human receptor analog (6′-sialyl-N-acetyllactosamine [6′SLN]). In addition, we also generated a variant NL219 HA lacking glycosylation at position 123 to investigate its role in the receptor binding properties of the NL219 HA. These results provide the structural basis for the receptor specificity of NL219 HA and rationalize differences in receptor binding in an effort to explain its increased replication efficiency and tissue distribution.



cDNA encoding for residues 1 to 497 of the mature HA ectodomain from A/Netherlands/219/2003 (H7N7) (NL219) was cloned into the baculovirus transfer vector, pAcGP67-A (BD Biosciences), in-frame with an N-terminal baculovirus GP67 signal peptide and a C-terminal thrombin cleavage site, a “foldon” sequence (18) and a hexahistidine tag at the extreme C terminus of the fusion protein to enable purification (46). An NL219 HA mutant (Thr125Ala) lacking a glycosylation motif at Asn123 was also generated by mutagenesis of the wild-type NL219 HA plasmid using the QuikChange multi-site-directed mutagenesis kit (Stratagene, La Jolla, CA). Transfection and virus amplification were carried out according to the baculovirus expression system manual (BD Biosciences, San Jose, CA).

Protein expression and purification.

Soluble HA ectodomain was purified from the culture supernatant by metal affinity chromatography and gel filtration chromatography and subjected to thrombin digestion as described previously (58). NL219 HA trimers were buffer exchanged into 10 mM Tris-HCl–50 mM NaCl (pH 8.0) and concentrated to 7 mg/ml for crystallization trials. At this stage, the HA protein contained additional plasmid-encoded residues at both the N (ADPG) and the C (SGRLVPR) termini.

Crystallization, ligand soaking, and data collection.

Initial nanoscale crystallization trials were set up using a Topaz free interface diffusion crystallizer system (Fluidigm Corp., San Francisco, CA). Crystals were observed in several conditions containing PEG polymers of various molecular weights. After optimization, diffraction quality crystals for NL219 HA were obtained at 20°C using a modified method for microbath under oil (9), by mixing the protein with a reservoir solution containing 20% PEG 8000 and 1 M lithium chloride. For receptor analog complexes, crystals were soaked for 1 h in the crystallization buffer containing 10 mM 3′SLN or 6′SLN (V-Labs, Inc., Covington, LA). All crystals were flash-cooled at 100K using 30% glycerol as the cryoprotectant. The data sets were collected at the Argonne National Laboratory Advanced Photon Source beamlines 22 ID and BM at 100K. The data were processed with the Denzo-Scalepack suite (35). The statistics on data collection are presented in Table 1.

Table 1
Data collection and refinement statistics

Structure determination and refinement.

The structure of NL219 was determined by molecular replacement with Phaser (30) using the structure of the avian H7 (Av-H7) from A/turkey/Italy/2002, PDB: 1TI8 (HA1, 99% identity; HA2, 98% identity) as a search model. One HA trimer occupies the asymmetric unit with an estimated solvent content of 62% based on a Matthews' coefficient (Vm) of 3.3 Å3/Da. Rigid body refinement of the trimer led to an overall R/Rfree of 39%/41%. The model was then “mutated” to the correct sequence, rebuilt by Coot (14) and then refined with REFMAC (6) using TLS refinement (56). The final model was assessed using MolProbity (10). The two complex structures were refined and evaluated using the same strategy. All statistics for data processing and refinement are presented in Table 1.

Glycan binding analyses.

Glycan microarray printing and recombinant HA analyses have been described previously (4, 4345, 57). Imprinted slides produced specifically for influenza research at the Centers for Disease Control and Prevention (CDC) using the Consortium for Functional Glycomics glycan library (CDC version 1 slides; see Table S1 in the supplemental material for glycans used in these experiments) were used. For kinetic studies, biotinylated glycans—Neu5Ac(α2-3)Gal(β1-4)Glc-biotin (3′SLN-b), Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcb-biotin (6′SLNLN-b), Neu5Ac(α2-6)Gal(β1-4)Glc-biotin (3′SLN-b), and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAcb-biotin (6′SLNLN-b)—obtained from the Consortium for Functional Glycomics ( through the resource request program, were coupled to streptavidin coated biosensors (Fortebio, Inc.). Recombinant HA was diluted to 4.42 μM trimer in kinetics buffer (phosphate-buffered saline containing 0.02% Tween 20, 0.005% sodium azide, and 100 μg of bovine serum albumin/ml). Binding was analyzed by biolayer interferometry (BLI) on an Octet Red instrument (Fortebio, Inc.) according to the manufacturer's instructions, and 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 NL219 HA are available from the RCSB Protein Data Bank (PDB) under accession codes 4DJ6 for the unliganded NL219 HA, 4DJ7 for the NL219 HA with 3′SLN, and 4DJ8 for NL219 HA with 6′SLN.


Overall structure of the HA.

The three-dimensional HA structure of the trimeric ectodomain from NL219 was determined by X-ray crystallography at 2.6-Å resolution (Table 1). In addition, two NL219 HA structures, complexed with an avian or human receptor analog (3′SLN or 6′SLN, respectively) were determined, both to a 2.8-Å resolution (Table 1). The overall structure is similar to those of previously reported HAs, with a globular head containing the receptor binding site and a vestigial esterase domain, and a membrane-proximal domain with its distinctive, central helical stalk, and HA1/HA2 cleavage site (21, 22, 28, 39, 41, 44, 45, 55, 57, 58). Six asparagine-linked glycosylation sites are predicted in the NL219 HA monomer but interpretable carbohydrate electron density was observed only at four sites: Asn28, Asn123, and Asn231 in HA1 and Asn82 in HA2 (Fig. 1A). At these sites, only one or two N-acetylglucosamines could be interpreted. HA 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 form. Highly pathogenic viruses, of which NL219 is an example, possess a polybasic cleavage site that can be cleaved by furin-like and many other serine proteases. Thus, in the baculovirus-infected insect cell expression system used for these studies, NL219 HA was produced in the HA1/HA2 form (Fig. 1A).

Fig 1
Structural overview of NL219 HA monomer. (A) One monomer is shown, with the HA1 chain colored in green and the HA2 chain in cyan. The locations of the glycosylation sites are labeled. (B) RBS of NL219 HA with the three structural elements comprising this ...

The 16 HA subtypes are divided into two groups based on phylogenic analysis (39). Group 1 can be further divided into three clades (H8, H9, and H12; H1, H2, H5, and H6; and H11, H13, and H16), and group 2 includes two clades (H3, H4, and H14; H7, H10, and H15) (41). Comparison of the NL219 HA monomer to other group 2 HA structures reveals a remarkable similarity. Superimpositions of the NL219 HA structure with avian H7 (PDB 1TI8), human H3 (PDB 2HMG), and avian H14 (PDB 3EYK) produced Cα atom root mean square deviations (RMSDs) of 0.62, 1.78, and 2.03 Å, respectively (39, 41, 52), whereas superimposition with human-H5 (PDB 2FK0) and 2009 pandemic H1 (PDB 3M6S) from group 1 gives Cα atom RMSDs of 3.52 and 3.27 Å, respectively (45, 57). Comparison of individual domains also yielded comparable results. (see Table S2 in the supplemental material).


The receptor binding site (RBS) is at the membrane distal end of each HA monomer (Fig. 1A), and its specificity for sialic acid and the vicinal galactose residue contributes to host range restriction. The consensus RBS in all influenza A HAs is composed of three structural elements: a 180-helix (residues 178 to 185), 210-loop (residues 211 to 219), and 120-loop (residues 123 to 128). In addition, the highly conserved residues Tyr88, Trp143, His174, and Tyr186—equivalent to Tyr98, Trp153, His183, and Tyr195 in H3 numbering (see Fig. S1 in the supplemental material)—form the base of the pocket (Fig. 1B). For H7 HAs, previous structural analyses also highlighted a two-amino-acid insertion in the nearby 140-loop that produces a >6-Å movement of this 140-loop toward the RBS (39, 40, 58). Although close to the RBS, there is currently no evidence that this loop contributes directly to receptor binding; modeling analysis does suggest that longer α2,6 receptor analogs may clash if they bind in a similar fashion as described for H1 and H3 HAs (40). Comparison of the RBS from NL219 with two previously published H7 HAs (PDB 1TI8 and PDB 3M5G) (39, 58) reveals an almost identical RBS (Fig. 1C). Interestingly, one additional glycosylation site observed at Asn123 of NL219 HA was absent in all other sequenced human isolates from the 2003 Netherlands outbreak or LPAI H7 from either Eurasia or North America. Asn123 is located within the equivalent of antigenic site A in H3 viruses, on the lower right-hand side of the RBS (54). Electron density for the asparagine-linked N-acetylglucosamine of the glycan at this site was observed in all three monomers of the trimeric HA, while the second N-acetylglucosamine was only visualized at chain A Asn123 (Fig. 1C).

NL219 HA-avian receptor analog complex.

To understand from a structural perspective how NL219 HA interacts with host receptors, we solved the structure of NL219 HA in complex with both avian and human analogs. For the avian receptor analog, 3′SLN, the electron density maps revealed well-ordered features for the SA-1, Gal-2, and GlcNAc-3 in the NL219 HA complex structure. Most of the hydrogen bonds were formed between SA-1 and residues (Tyr88, Thr125, Thr126, Ser127, His174, Glu181, and Gln217) within the pocket (Fig. 2A). Residue Gln217 (226 in H3 numbering; see Fig. S1 in the supplemental material), one of the key residues in receptor specificity and host adaptation, also binds to Gal-2 in 3′SLN. No hydrogen bonding was apparent between the HA and GlcNAc-3.

Fig 2
Binding of the avian receptor analog to the NL219 RBS. (A) RBS of NL219 with 3′SLN into the pocket. Putative hydrogen bond interactions between the glycan and the HA residues, calculated by hbplus (31), are shown as black broken lines. (B) Overlap ...

Structural comparison of NL219 HA/3′SLN to other published HA complexes, including human H1 (PDB 1RVX) (18), avian H2 (PDB 2WR2) (28), avian H3 (PDB 1MQM) (21), avian H5 (PDB 1JSN) (21), avian H7 (40), and swine H9 (PDB 1JSH) (22) subtypes, revealed high similarity (Fig. 2B). The trans conformation of the α2-3 linkage in the avian receptor analog points out of the RBS and the terminal SA-1 is positioned almost identically in all structures. The Gal-2 also remains in very close proximity to its counterpart in other structural complexes. The NL219 and all previously reported complexes with α2-3 linked analogs revealed no hydrogen bonding between the HA and GlcNAc-3, which suggests that for α2-3 linkage glycans, only the first two saccharides are required for HAs to bind to avian receptors (19, 22, 28, 58).

NL219 HA-human receptor analog complex.

In the NL219 HA/6′SLN complex, there was only interpretable electron density for the SA-1 of the 6′SLN. Although the SA-1 still forms a hydrogen bonding network with HA, fewer interactions were found compared to the 3′SLN complex structure (Fig. 3A). The terminal SA-1 remains in the same position in the NL219 HA/6′SLN complex structure as other published HA-receptor complexes, including human seasonal H1 (PDB 1RVZ) (19), avian H2 (PDB 2WR1) (28), avian H3 (PDB 1MQN) (21), avian H5 (PDB 1JSO) (21), and swine H9 (PDB 1JSI) (21) (Fig. 3B). The cis conformation of the α2-6 linkage in human receptor analogs has a “turn” between the first and second position of the analogs. The superimposed crystal structures show significant variation at Gal-2. Human seasonal H1 HAs form direct hydrogen bonds between both Lys222 and Asp225 (H3 numbering) to the Gal-2 (19), whereas avian H2 HA uses water molecules to mediate the binding between both Lys222 and Asp225 (H3 numbering) to the Gal-2 (28). Swine-H9 HA has only one hydrogen bond between Leu226 (H3 numbering) and Gal-2, while receptor analog complexes of H5 and H7 HAs, as seen here, usually only have the SA-1 interpretable in the electron density map (22, 58). Clearly, the cis conformation of α2-6 SA-Gal linkage in human receptor is not favored by NL219 or other HA molecules of avian origin.

Fig 3
Binding of the human receptor analog to the NL219 RBS. (A) RBS of NL219 with 6′SLN into the pocket. Putative hydrogen bond interactions between the glycan and the HA residues, calculated by hbplus (31), are shown as black broken lines. (B) Overlap ...

Glycan array and kinetics.

Previous studies reported that when an Ala125Thr substitution, which generates a glycosylation site at Asn123 near the RBS, was introduced into the HA of an HPAI H7N7 virus isolated from a clinically mild infection, the resulting virus showed increased replication efficiency and tissue distribution (13, 32). In order to assess the effect of this substitution on receptor binding, we expressed and purified NL219 protein containing a Thr125Ala mutation (NL219ΔCHO) that removed the glycosylation site at position 123 and subjected both recombinant HAs to glycan microarray analysis. The results for wild-type NL219 revealed no binding to any of the α2-6-linked sialosides present on the array, while there was a strong binding preference for the majority of α2-3-linked sialosides (Fig. 4A and see Table S1 in the supplemental material). In contrast, recombinant NL219ΔCHO HA resulted in a more restricted binding profile (Fig. 4B and see Table S1 in the supplemental material). Only 7 of the 31 glycans (glycans 6, 9 to 11, 60, 61, and 63) to which WT NL219 bound, giving fluorescent signals >30,000 (comprising a sulfated α2-3 sialoside, as well as α2-3 and α2-3/α2-6 disialosides), bound to a comparable level with the mutant (see Table S1 in the supplemental material). The remaining 24 glycans all had significantly reduced binding signals, with 13 of these glycans (i.e., glycans 4, 12, 15, 17, 18, 21, 25, 26, 28, 29, 32, 33, and 62) being considered nonbinders.

Fig 4
Glycan microarray and kinetic binding analysis of wild-type and mutant NL219 and NL219ΔCHO recombinant HAs. To assess the effect of HA1 Asn123 glycosylation on receptor specificity, NL219 (A) and NL219ΔCHO (B) recombinant HAs were analyzed ...

Glycan binding to recombinant protein was further analyzed by BLI using an Octet Red system from ForteBio, Inc. This label-free technology was used to measure recombinant HA binding to biotinylated glycans (3′SLN-b, 3′SLNLN-b, 6′SLN-b, and 6′SLNLN-b) preloaded onto streptavidin-coated biosensors. The results for both proteins confirm glycan microarray results in that only α2-3-linked analogs bound (Fig. 4C and andD).D). In addition, the weaker binding signals for the NL219ΔCHO protein were confirmed by the kinetic results such that the wild-type NL219 recombinant HA had five to seven times higher association rates (kon) for both 3′SLN-b and 3′SLNLN-b (with apparent KDs of 0.5 and 0.6 μM, respectively) than to NL219ΔCHO (with apparent KDs of 2.1 and 3.0 μM, respectively) (Table 2).

Table 2
Kinetic results for glycan binding to NL219 and NL219ΔCHOa


Subtype H7 influenza viruses continue to circulate across Europe and North America and, since 2002, more than 100 human H7 subtype infections have been reported. Thus, H7 influenza viruses continue to be a public health concern, and little is known about the exact molecular determinants required for efficient interspecies transmission and pathogenicity. One critical molecule, however, is the viral coat protein HA, a key molecule whose receptor is responsible for viral attachment and entry.

Previous structural studies of HAs and complexes with receptor analogs have provided considerable insight into receptor binding interactions (19, 21, 22, 28, 58). We report here the trimeric NL219 HA structure, from a fatal human infection in the course of the 2003 H7N7 poultry outbreak in the Netherlands. The overall structure of NL219 HA is very similar to other avian H7 HAs (Fig. 1C) and the group 2 HAs in general. Our structural analyses of NL219 HA complexes with both avian and human receptor analogs confirms that the NL219 RBS favors binding to only avian α2-3 linked receptor analogs. Although electron density maps revealed only the sialic acid in the NL219/6′SLN structure, well-ordered features for all of the components of the 3′SLN analog (SA-1, Gal-2, and GlcNAc-3) were visible, helped by additional hydrogen bonding by Gln217 (226 in H3 numbering) of NL219 to the Gal-2 (Fig. 2). This is in agreement with previous virus glycan array data (2, 58).

Previous studies have attempted to explain at the molecular level why the infection with NL219 virus was lethal, while those with other HPAI H7N7 viruses, such as NL33 and NL230 from the same outbreak, were not (3, 13, 17, 32). Comparison of the NL219 whole-genome sequence to nonfatal virus isolates, NL230 and NL33, revealed a number of amino acid substitutions, of which three were in the mature HA protein (Table 3) (3, 17). Of these HA differences, an NL219 Ala125Thr substitution that introduced an N-linked glycosylation site at Asn123 was reported to increase replication efficiency and modulate tissue distribution of the NL219 virus (13, 32). In particular, differences between NL219 and a nonlethal virus from the same outbreak were observed in the lower respiratory tract where NL219 attached more abundantly to nonciliated cuboidal cells in the bronchioles, alveolar macrophages, and type 2 pneumocytes in the alveoli (32). It was suggested that receptor specificity might be affected (13), although this was not apparent when NL219 and NL230 viruses were compared by glycan microarray analysis (2).

Table 3
Amino acid differences between lethal (NL219) and other nonlethal H7N7 viruses from the same outbreak

It is important to note that insect cells differ from mammalian cells in that they do not produce complex glycans, although high-mannose glycans can be produced (26). Due to the presence of NA, complex glycans attached to influenza HAs from mammalian cells usually terminate in galactose. Despite these differences, previously published results for glycan microarrays comparing recHA to virus binding show good agreement for high-affinity interactions (44, 58), suggesting that the sizes of the N-glycans from insect cells approximates those of complex N-glycans produced in mammalian host cells. However, the closer a glycosylation site is to the RBS, the greater potential it has for affecting receptor binding through steric hindrance, as shown by Wang et al., who reported that the systematic simplification of N-glycans on an H5 HA resulted in an increase in binding to α2,3 sialosides (50).

From our structural analyses, it is clear that the glycosylation site at Asn123 is occupied in our insect cell-expressed HA due to the clear electron density for the first one or two N-acetylglucosamines in the glycan structure (Fig. 1C). Thus, this recombinant HA can be used to characterize the effect of glycosylation at this position on receptor binding. In addition to the wild-type protein, we also expressed NL219ΔCHO with a Thr125Ala mutation to remove this glycosylation site and subjected both recombinant HAs to glycan microarray analysis. The results for wild-type recombinant HA were comparable to the results published for the NL219 virus (2, 58). Whereas the wild-type HA possessed a strong and broad binding preference for most α2-3 glycans on the array, the Thr125Ala mutant revealed a much weaker binding profile. This weak binding was also confirmed by kinetic analysis using a biosensor assay that detects real-time binding of the recombinant HA to biotinylated glycans (Fig. 4C and andDD and Table 3). Ohuchi et al. studied the fowl plague virus (FPV), an H7 virus with an equivalent glycosylation site to NL219 at position 123, plus an additional site at 149 (34). Although the loss of glycosylation at position 123 reduced binding affinity on the NL219 backbone, the authors of that study showed that the elimination of both 123 and 149 sites increased affinity of the FPV virus for its receptor, and although position 149 was the dominant regulator, position 123 still had an effect.

However, the results presented here highlight a potential mechanism that might explain other reported observations (13, 32). Rather than switching receptor specificity to a preference for human type receptors, as suggested elsewhere, Asn123 glycosylation appears to improve binding affinity to α2-3 receptor analogs, in agreement with viral data reporting better NL219 binding in the lower respiratory tract (32). Why glycosylation at Asn123 improves binding to avian type receptors cannot be fully explained by these structures. However, our results indicate that the glycan points away from the RBS (Fig. 2A and and3A)3A) and appears unlikely to interact directly with the bound glycan. Thus, two possibilities exist to explain the increase in α2-3 binding. First, one unique feature of the H7 RBS is the 140-loop that has a two-amino-acid insertion, producing more than 6-Å movement of the loop toward the RBS (Fig. 1C) (39, 58). The stability of this loop and its interaction with the 180-helix might be enhanced by the introduction of the glycosylation site. Comparison of the RBS of the avian A/turkey/Italy/2002 (Tk02) H7 HA (PDB 1TI8) to the NL219 structure highlights Arg131, which interacts with the unoccupied Asn123 in the Tk02 structure, but shifts upward to interact with the main chain carbonyls of Ser145 and Asn146 in the 140-loop of the NL219 structure (Fig. 5). Second, compared to other viruses from the 2003 outbreak, the NL219 Asn123 glycosylation site was introduced indirectly by an Ala125Thr mutation. Position 125 is in the region where the 120-loop interacts directly with the sialic acid, and although the side chain of Thr125 cannot interact directly with the sialic acid, it could improve loop stability and receptor affinity by contributing to the hydrogen bonding network in this region.

Fig 5
Movement of Arg131 near the Asn123 glycosylation site. The RBS overlaps of NL219 (green) and Tk02-H7 (magenta) are shown in the diagram. Residues mentioned in the main text are labeled and shown as sticks. The major structural elements of the RBS are ...

Although HPAI viruses with the H5 subtype HA in combination with several subtypes of NA have caused many influenza outbreaks among poultry, reported infections in humans are exclusively of the H5N1 subtype. In contrast, H7 HAs in combination with several other NA subtypes have been successfully transmitted from birds to humans, which suggests a broad compatibility between H7 subtype and multiple NA subtypes. In addition, the conjunctival tropism of H7 subtype infections is unlike infections with other subtypes. Therefore, H7 subtype viruses have unique properties that require further research to support the development of preventive interventions. The identification of mutations that enhance virulence also underlines the need for continuous virus surveillance and risk assessment.

Supplementary Material

Supplemental material:


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.

We also thank the staff of SER-CAT sector 22 for their help for data collection. We thank WHO Global Influenza Surveillance and Response System (GISRS) for providing the NL219 virus and Limei Chen (CDC) for kindly providing the NL219 gene used to generate the baculovirus clones described in this study. 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. Glycan microarray data presented here will be made available on-line through the CFG web site upon publication ( We also thank the CFG for the supplying glycans for direct binding experiments through their resource request program. For analyses, we thank Rupert Russell (St. Andews University) for kindly providing the structure coordinate file for the avian H7/3′SLN complex.

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.


Published ahead of print 6 June 2012

Supplemental material for this article may be found at


1. Banks J, Speidel E, Alexander DJ. 1998. Characterisation of an avian influenza A virus isolated from a human: is an intermediate host necessary for the emergence of pandemic influenza viruses? Arch. Virol. 143:781–787 [PubMed]
2. Belser JA, et al. 2008. Contemporary North American influenza H7 viruses possess human receptor specificity: implications for virus transmissibility. Proc. Natl. Acad. Sci. U. S. A. 105:7558–7563 [PubMed]
3. Belser JA, et al. 2007. Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans. J. Virol. 81:11139–11147 [PMC free article] [PubMed]
4. Blixt O, et al. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033–17038 [PubMed]
5. Brown IH. 2010. Summary of avian influenza activity in Europe, Asia, and Africa, 2006–2009. Avian Dis. 54:187–193 [PubMed]
6. CCP4 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50:760–763 [PubMed]
7. Centers for Disease Control and Prevention 2004. Update: influenza activity–United States and worldwide, 2003-04 season, and composition of the 2004-05 influenza vaccine. MMWR Morb. Mortal. Wkly. Rep. 53:547–552 [PubMed]
8. Centers for Disease Control and Prevention 2004. Update: influenza activity–United States, 2003-04 season. MMWR Morb. Mortal. Wkly. Rep. 53:284–287 [PubMed]
9. Chayen NE, Shaw-Steward PD, Blow DM. 1992. Microbatch crystallization under oil: a new technique allowing many small volume crystallization experiments J. Crystallogr. Growth 122:176–180
10. Davis IW, et al. 2007. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35:W375–W383 [PMC free article] [PubMed]
11. de Jong JC, et al. 2003. The 2002/2003 influenza season in the Netherlands and the vaccine composition for the 2003/2004 season. Ned. Tijdschr. Geneeskd. 147:1971–1975 [PubMed]
12. DeLano WL. 2002. The PyMOL molecular graphics systems, v1.5.0.4 Schrödinger, LLC, Cambridge, MA:
13. de Wit E, et al. 2010. Molecular determinants of adaptation of highly pathogenic avian influenza H7N7 viruses to efficient replication in the human host. J. Virol. 84:1597–1606 [PMC free article] [PubMed]
14. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60:2126–2132 [PubMed]
15. Eurosurveillance 2007. Avian influenza A/(H7N2) outbreak in the United Kingdom. Euro Surveill. 12:E070531.2 [PubMed]
16. Fouchier RA, et al. 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79:2814–2822 [PMC free article] [PubMed]
17. Fouchier RA, et al. 2004. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc. Natl. Acad. Sci. U. S. A. 101:1356–1361 [PubMed]
18. Frank S, et al. 2001. Stabilization of short collagen-like triple helices by protein engineering. J. Mol. Biol. 308:1081–1089 [PubMed]
19. Gamblin SJ, et al. 2004. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303:1838–1842 [PubMed]
20. Garten RJ, et al. 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325:197–201 [PMC free article] [PubMed]
21. Ha Y, Stevens DJ, Skehel JJ, Wiley DC. 2003. X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus. Virology 309:209–218 [PubMed]
22. Ha Y, Stevens DJ, Skehel JJ, Wiley DC. 2001. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. U. S. A. 98:11181–11186 [PubMed]
23. Hirst M, et al. 2004. Novel avian influenza H7N3 strain outbreak, British Columbia. Emerg. Infect. Dis. 10:2192–2195 [PMC free article] [PubMed]
24. Kawaoka Y, Bean WJ, Webster RG. 1989. Evolution of the hemagglutinin of equine H3 influenza viruses. Virology 169:283–292 [PubMed]
25. Koopmans M, et al. 2004. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 363:587–593 [PubMed]
26. Kost TA, Condreay JP, Jarvis DL. 2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol. 23:567–575 [PMC free article] [PubMed]
27. Kurtz J, Manvell RJ, Banks J. 1996. Avian influenza virus isolated from a woman with conjunctivitis. Lancet 348:901–902 [PubMed]
28. Liu J, et al. 2009. Structures of receptor complexes formed by hemagglutinins from the Asian influenza pandemic of 1957. Proc. Natl. Acad. Sci. U. S. A. 106:17175–17180 [PubMed]
29. Matrosovich MN, et al. 1997. Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology 233:224–234 [PubMed]
30. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ. 2005. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61:458–464 [PubMed]
31. McDonald IK, Thornton JM. 1994. Satisfying hydrogen bonding potential in proteins., J. Mol. Biol. 238:777–793 [PubMed]
32. Munster VJ, et al. 2007. The molecular basis of the pathogenicity of the Dutch highly pathogenic human influenza A H7N7 viruses. J. Infect. Dis. 196:258–265 [PubMed]
33. Nguyen-Van-Tam JS, et al. 2006. Outbreak of low pathogenicity H7N3 avian influenza in UK, including associated case of human conjunctivitis. Euro Surveill. 11:E060504.2. [PubMed]
34. Ohuchi M, Ohuchi R, Feldmann A, Klenk HD. 1997. Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J. Virol. 71:8377–8384 [PMC free article] [PubMed]
35. Otwinowski A, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307–326
36. Peiris M, et al. 1999. Human infection with influenza H9N2. Lancet 354:916–917 [PubMed]
37. Puzelli S, et al. 2005. Serological analysis of serum samples from humans exposed to avian H7 influenza viruses in Italy between 1999 and 2003. J. Infect. Dis. 192:1318–1322 [PubMed]
38. Rogers GN, et al. 1983. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304:76–78 [PubMed]
39. Russell RJ, et al. 2004. H1 and H7 influenza haemagglutinin structures extend a structural classification of haemagglutinin subtypes. Virology 325:287–296 [PubMed]
40. Russell RJ, Stevens DJ, Haire LF, Gamblin SJ, Skehel JJ. 2006. Avian and human receptor binding by hemagglutinins of influenza A viruses. Glycoconj. J. 23:85–92 [PubMed]
41. Russell RJ, et al. 2008. Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion. Proc. Natl. Acad. Sci. U. S. A. 105:17736–17741 [PubMed]
42. Scholtissek C, Rohde W, Von Hoyningen V, Rott R. 1978. On the origin of the human influenza virus subtypes H2N2 and H3N2. Virology 87:13–20 [PubMed]
43. Stevens J, et al. 2008. Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J. Mol. Biol. 381:1382–1394 [PMC free article] [PubMed]
44. Stevens J, et al. 2006. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. 355:1143–1155 [PubMed]
45. Stevens J, et al. 2006. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312:404–410 [PubMed]
46. Stevens J, et al. 2004. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303:1866–1870 [PubMed]
47. Subbarao K, et al. 1998. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279:393–396 [PubMed]
48. Thompson MG, et al. 2010. Estimates of deaths associated with seasonal influenza–United States, 1976–2007. MMWR Morb. Mortal. Wkly. Rep. 59:1057–1062 [PubMed]
49. Tweed SA, et al. 2004. Human illness from avian influenza H7N3, British Columbia. Emerg. Infect. Dis. 10:2196–2199 [PMC free article] [PubMed]
50. Wang C, et al. 2009. Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc. Natl. Acad. Sci. U. S. A. [PubMed]
51. Webster RG, Geraci J, Petursson G, Skirnisson K. 1981. Conjunctivitis in human beings caused by influenza A virus of seals. N. Engl. J. Med. 304:911. [PubMed]
52. Weis WI, Brunger AT, Skehel JJ, Wiley DC. 1990. Refinement of the influenza virus hemagglutinin by simulated annealing. J. Mol. Biol. 212:737–761 [PubMed]
53. World Health Organization 1980. A revision of the system of nomenclature for influenza viruses: a WHO memorandum. Bull. World Health Organ. 58:585–591 [PubMed]
54. Wiley DC, Wilson IA, Skehel JJ. 1981. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289:373–378 [PubMed]
55. Wilson IA, Skehel JJ, Wiley DC. 1981. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289:366–373 [PubMed]
56. Winn MD, Isupov MN, Murshudov GN. 2001. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57:122–133 [PubMed]
57. Yang H, Carney P, Stevens J. 2010. Structure and receptor binding properties of a pandemic H1N1 virus hemagglutinin. PLoS Curr. 2:RRN1152. [PMC free article] [PubMed]
58. Yang H, Chen LM, Carney PJ, Donis RO, Stevens J. 2010. Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site. PLoS Pathog. 6:e1001081 doi:10.1371/journal.ppat.1001081 [PMC free article] [PubMed]

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