Structural basis for receptor specificity of influenza B virus hemagglutinin

Abstract

Receptor-binding specificity of HA, the major surface glycoprotein of influenza virus, primarily determines the host ranges that the virus can infect. Influenza type B virus almost exclusively infects humans and contributes to the annual “flu” sickness. Here we report the structures of influenza B virus HA in complex with human and avian receptor analogs, respectively. These structures provide a structural basis for the different receptor-binding properties of influenza A and B virus HA molecules and for the ability of influenza B virus HA to distinguish human and avian receptors. The structure of influenza B virus HA with avian receptor analog also reveals how mutations in the region of residues 194 to 196, which are frequently observed in egg-adapted and naturally occurring variants, directly affect the receptor binding of the resultant virus strains. Furthermore, these structures of influenza B virus HA are compared with known structures of influenza A virus HAs, which suggests the role of the residue at 222 as a key and likely a universal determinant for the different binding modes of human receptor analogs by different HA molecules.

Influenza virus, including type A and type B, remains a major cause of morbidity and mortality in humans. In a typical influenza infection, the first close contact with the host cell is the binding of the virus to its cellular receptors, terminal sialic acids of glycoproteins and glycolipids (1, 2). This binding is mediated by HA, a major surface glycoprotein of influenza A or B virus.

Influenza A virus infects a wide variety of hosts including humans, avians, equines, and swines. The HA molecules of influenza A viruses isolated from different hosts differ in their ability to recognize different cellular receptors in which the linkage between sialic acid and galactose of the carbohydrate chain is either α(2,3) or α(2,6). HA molecules of human viruses preferentially bind to α(2,6)-linked receptors, those of avian and equine viruses bind to α(2,3)-linked, and those of swine viruses bind to both (1–9). Thus, α(2,3)-linked and α(2,6)-linked receptors are also referred to as avian and human receptors, respectively. X-ray crystallographic studies of influenza A virus HAs have defined the receptor-binding site as a shallow depression at the top of the molecule, formed by a set of conserved residues (10–19). We now have a detailed understanding of the molecular determinants for the different receptor specificity of different subtypes of influenza A virus HAs, in particular for H1, H2, and H3 subtypes that infect humans (1–3, 10–28). For H2 and H3 HAs, it is predominantly determined by residues at 226 and 228 (Leu-226 and Ser-228 in human HAs and Gln-226 and Gly-228 in avian HAs, following the numbering convention for H3 HA) (5, 7, 11, 21, 29–31). However, H1 HAs are different in this regard: although human and avian H1 HAs recognize α(2,6)-linked or α(2,3)-linked receptors, respectively, they both have Gln-226 and Gly-228. Instead, residues at other sites appear to determine the receptor specificity (8, 16, 28, 32). For instance, mutations at residues 190 and 225 in human 1918 H1 HA completely changed the receptor specificities (28, 32). The different receptor specificity of HAs appears to be a major barrier for cross-species transmission (2). This justifies the ongoing global efforts to identify mutations on avian H5N1 HA that would render the virus the ability to rapidly infect humans (18, 25–27). Such knowledge will allow prompt actions to stop H5N1 pandemics as soon as a pandemic strain emerges.

Despite the obvious significance of influenza B virus in epidemiology (33), in sharp contrast to our extensive knowledge on the receptor-binding specificity of influenza A virus HAs, very little is known about influenza B virus HA from a structural standpoint (34). For example, it was unknown how the binding modes of influenza B virus HA with its cellular receptors differ from those of influenza A virus HAs, particularly in the light of the rather low sequence identity (≈25%) between them (35, 36). Moreover, it was unclear whether the HA of influenza B virus, which circulates exclusively in humans, preferentially binds to α(2,6)-linked receptors as human influenza A virus HAs or binds equally well to α(2,3)-linked and α(2,6)-linked receptors (6, 37–41). This was further complicated by the probability of altered receptor specificity due to egg adaptation of cultured viruses (6, 40). For instance, influenza B virus HA tends to lose its glycosylation at residue 194 when grown in embryonated chicken eggs as a result of the mutations at residues 194 or 196 (42). Those mutant HAs apparently have an augmented affinity for α(2,3)-linked receptors that are abundant in chicken eggs (40). However, it remained to be determined whether the mutations themselves, or the loss of glycosylation, is responsible for such changes in affinity.

To address these unresolved issues, we have determined the crystal structures of HA of the influenza B/HongKong/8/73 strain (hereafter referred to as influenza B/HK HA) in complex with α(2,3)-linked and α(2,6)-linked receptor analogs, respectively. These structures provide a detailed understanding of the interactions by which influenza B virus HA distinguishes human and avian receptors. To elucidate the structural basis for the differences in receptor specificity between influenza A and B virus HAs, the structures of influenza B/HK HA are compared with those of human influenza A virus H3 HA (X31), the most extensively studied HA molecule (10–14). Furthermore, by comparing the structure of influenza B/HK HA with a number of known structures of influenza A virus HAs complexed with α(2,6)-linked human receptor analogs, we propose an important role of the residue at 222 as likely a universal determinant for the different binding modes of human receptor analogs by different types and subtypes of HA molecules.

Results

Receptor-Binding Site of Influenza B/HK HA.

The complex structures of influenza B/HK HA with α(2,3)-linked and α(2,6)-linked receptor analogs were determined to 2.8-Å resolution with one subunit per asymmetric unit (Table 1). The final model for each subunit contains HA1 1 to 342 and HA2 1 to 169. Despite the relatively low sequence identity between them, ≈20% for HA1 and 30% for HA2, influenza B/HK HA shares an overall similar fold to influenza A virus HAs (1, 2, 10–19). It has an elongated membrane-proximal domain and a globular membrane-distal domain (Fig. 1a). The biological unit of HA is a homotrimer of HA1/HA2 subunits (Fig. 1a). The receptor-binding site is located at the top of the membrane-distal domain (Fig. 1a) formed by 190-helix (HA1 193 to 202) at the top, 240-loop (HA1 237 to 242) as the left edge, and 140-loop (HA1 136 to 143) as the right edge (Fig. 1b). Four residues from the HA1 chain, Phe-95, Trp-158, His-191, and Tyr-202, constitute the base of the receptor-binding site, all of which are absolutely conserved among all known sequences of influenza B virus HAs (35, 36, 43–50). In addition to π-stacking interactions among these four aromatic residues, the receptor-binding site is further stabilized by a hydrogen bond between His-191 and Tyr-202 and four more hydrogen bonds between Asp-193 and Ser-240 (Fig. 1b). The latter interactions make the left entrance of the binding site narrower by 0.3 Å (Fig. 1b) than that of human H3 HA (between Glu-190 and Ser-228).

Table 1.

Crystallographic statistics of B/HK HA–receptor complex structures

	LSTa	LSTc
Data collection statistics
Space group	P321	P321
Unit cell, Å	a = b = 98.33, c = 135.99	a = b = 98.37, c = 135.90
No. of unique reflections	19,145	19,705
Resolution, Å	13–2.7	20–2.7
Completeness, %	89.9 (87.6)	92.6 (89.9)
I/σI	16.6 (2.1)	20.9 (2.6)
Rmerge, %	4.7 (29.1)	5.3 (29.7)
Refinement statistics
Resolution, Å	10–2.8	20–2.8
R/Rfree, %	27.99/29.83	29.48/31.07
rms bond length, Å	0.008	0.008
rms bond angle, °	1.48	1.52

Numbers in parentheses are for the highest-resolution shell.

Fig. 1.

Structure and receptor-binding site of influenza B/HK HA. (a) The overall structure of B/HK HA with three subunits differently colored. There is one receptor-binding site in each subunit at the distal end of molecule (away from the membrane), highlighted in red for clarity. (b) Close-up view of the receptor-binding site of B/HK HA. Important residues forming the receptor-binding site are explicitly drawn, with hydrogen-bonding interactions among them shown as dashed lines. (c) Comparison of the receptor-binding sites of B/HK (red) and human H3 (gray) HAs. Their corresponding residues are shown in yellow and cyan, respectively. Two large structural shifts between them, at residues Ser-140 and Asp-193, respectively, are indicated by black dashed lines with the distances labeled. The hydrogen-bonding interactions are shown as dashed lines.

One unique feature of the receptor-binding site of influenza B virus HA is a phenylalanine residue at position 95, which is in sharp contrast to a tyrosine residue for all influenza A virus HAs at equivalent position (Fig. 1c). This difference could be at least partially responsible for the different shape of the receptor-binding site and the different orientation of Trp-158 of influenza B virus HAs (Fig. 1c). Moreover, this subsite of B/HK HA binding site is further reshaped by the one-residue deletion after His-191 and a substitution of a proline residue by Ser-192 (41) (Fig. 1c). The structural difference of this subsite can be appreciated by a shift of 3.6 Å between the Cα atoms of Asp-193 in B/HK HA and its equivalent residue, Ser-186, in H3 HA (Fig. 1c).

In the region of 190-helix, the receptor-binding sites of B/HK HA and H3 HA are very similar. As previously suggested based on primary sequence alignment (41), the right edge of B/HK HA binding site is also very similar to that of human H3 HA and provides the conserved main-chain and side-chain atoms for interactions with the sialic acid moiety (Sia-1) of the receptors. These atoms include the main-chain carbonyl of residue Thr-139, the side-chain hydroxyl of Ser-140, and the main-chain amide and carbonyl of Gly-141 (equivalent to Gly-135, Ser-136, and Asn-137 in H3 HA, respectively). However, despite the conserved interactions, the residue Ser-140 of B/HK HA is located by ≈1.3 Å further away from the 240-loop than that of human H3 HA (Fig. 1c).

The left edge of the B/HK HA receptor-binding site harbors residues Pro-238 and Ser-240, which are at equivalent positions to residues 226 and 228 in H3 HA, respectively. Both residues 226 and 228 of H3 HA have been implicated as major determinants for receptor specificity (1, 2). Because of a four-residue insertion (residues 233 to 236) proceeding Leu-237 in influenza B/HK HA, this part of the receptor-binding site is markedly different from that of human H3 HA (Fig. 1c). The four-residue expansion results in much looser packing between the 240-loop and its proceeding loop (residues 230 to 236). Thus, to compensate for the interactions provided by Trp-222 in human H3 HA (see later for more discussion of its functional role), the side chain of Leu-237 in B/HK HA is flipped over to be located similarly to Trp-222 (Fig. 1c). The highly conserved residue Ser-240 is located by ≈3.6 Å higher than its equivalent residue, Ser-228, in human H3 HA. Although the side chain of Pro-238 is shorter than that of Leu-226 in H3 HA, the main chains of the 240-loop in B/HK HA are shifted toward the 140-loop so that the side-chain atoms responsible for interacting with the receptors are similarly located. Together with the ≈1.3-Å shift of the 140-loop in B/HK HA, the lower opening of the B/HK HA binding site (between Ser-140 and Pro-238) is ≈1.4 Å wider than that of human H3 HA (between Ser-136 and Leu-226). These structural differences between the receptor-binding sites of influenza B virus and human H3 HAs may account for the observed difference in receptor affinity and specificity between influenza A and B viruses (41).

Binding of Human Receptor Analog by Influenza B Virus HA.

α(2,6)-linked sialopentasaccharide LSTc, lactoseries tetrasaccharide c, was used as a human receptor analog (14) to study the interactions with influenza B/HK HA (Fig. 2a). Between Sia-1 and B/HK HA, there are two hydrogen bonds made by the main-chain amide and carbonyl of Gly-141, two hydrogen bonds by the side-chain hydroxyl of Ser-140, and two hydrogen bonds by the main-chain carbonyl of Thr-139 (Fig. 2a). On the other side of Sia-1, each of the side chains of residues Asp-193 and Ser-240 contributes one hydrogen bond with the 9-hydroxyl group of Sia-1. Except in only one case for Gly-141 (44, 45), all of those residues that interact with the Sia-1 moiety are absolutely conserved among different field isolates of influenza B viruses (35, 36, 43–50), highlighting their importance in receptor binding for viral infection.

Fig. 2.

Human receptor complexes. (a) The interaction of human receptor analogs in the receptor-binding site of B/HK HA. The hydrogen-bonding interactions are shown as dashed lines in cyan. The asialo portion of the receptors refers to the sugar rings other than Sia-1. (b) The interaction of human receptor analogs in the receptor-binding site of human H3 HA. The hydrogen-bonding interactions are shown as dashed lines in cyan. It is clear that the receptor-binding site of human H3 HA has an overall stronger interaction network with the bound human receptor analog than B/HK HA does. (c) Comparison of interactions of B/HK and human H3 HAs with the Sia-1 moiety. The ≈25° tilting of the Sia-1 ring around the C5–O6 axis is indicated by an arrow. The up-shift of the glycerol moiety of the Sia-1 in B/HK HA is also labeled. (d) The superposition of B/HK (green), human H3 (yellow), and swine H1 (purple) HAs in complex with human receptor analogs. It is clear that the interactions of Gal-2 ring with the residue at 222 affect the conformations of bound human analogs.

To compare, we show the human H3 HA–human receptor complex in Fig. 2b (11). In contrast to the total of six hydrogen bonds between Sia-1 of LSTc and 140-loop of B/HK HA, five hydrogen bonds are found between Sia-1 and 130-loop of human H3 HA, among which the new hydrogen bond between the side chain of Asn-137 and Sia-1 of LSTc is at a distance of 2.7 Å. Moreover, the glycerol moiety of Sia-1 makes more extensive interactions with the binding site of H3 HA. There are two hydrogen bonds with the glycerol moiety of Sia-1 by the hydroxyl of the tryosine residue (Tyr-98) and three additional hydrogen bonds by the side chains of Glu-190 and Ser-228 (Fig. 2b). The overall less extensive interactions between the Sia-1 moiety and B/HK HA provide an explanation for the experimental observation that influenza B viruses in general have lower binding affinity for Neu5Ac (representing only the Sia-1 moiety) than influenza A H1 and H3 viruses (41). It appears that the interactions with the receptor-binding site, in particular those made by Tyr-98, pull the ring of Sia-1 down deeper into the pocket in the case of influenza A HAs (Fig. 2c). In contrast, in B/HK HA where a Phe-95 is present, the glycerol moiety of Sia-1 exclusively hydrogen-bonds with the side chains of Asp-193 and Ser-240. The higher position of residue Ser-240 in the structure, in the absence of any hydrogen-bonding interaction from the base of the binding site, pulls up the glycerol side of Sia-1. Consequently, the glycerol moiety of Sia-1 in B/HK HA is ≈1.8 Å higher than that in human H3 HA, with an ≈25° tilting of the Sia-1 ring around the C5–O6 axis (Fig. 2c).

In addition to the Sia-1 moiety, Gal-2 and GlcNAc-3 of LSTc are clearly visible in the B/HK HA–LSTc complex (Fig. 2a). However, only fragmented density is observed for the terminal Gal-4 and Glc-5, which are presumably disordered. The majority of the interactions between LSTc and HA is made by the Sia-1 moiety, whereas the asialo portion of LSTc does not make significant contacts with B/HK HA, except for a relatively weak hydrogen bond between Gal-2 and the main-chain carbonyl of Leu-237 (at a distance of 3.9 Å) (Fig. 2a). The overall conformation of bound LSTc in B/HK HA is the most similar to that of LSTc in the complex with human H3 HA. Thus, if it follows the path of LSTc in human H3 HA complex (Fig. 2b), LSTc in B/HK HA would exit the receptor-binding site from the right-hand side of 190-helix (Fig. 2 a and b).

Binding of Avian Receptor Analog by Influenza B Virus HA.

α(2,3)-linked sialopentasaccharide LSTa, lactoseries tetrasaccharide a, was used as an avian receptor analog (14) to study the interactions with influenza B/HK HA (Fig. 3a). The full-length LSTa is clearly visible (Fig. 3a) in the complex structure. Because it is sitting 0.4–0.8 Å higher in the binding site than that of LSTc, the Sia-1 moiety of LSTa makes two fewer hydrogen bonds with B/HK HA that are contributed by the main-chain carbonyl of Thr-139 and the main-chain amide of Gly-141, respectively (Fig. 2a). Besides the Sia-1 moiety, Gal-2, GlcNAc-3, and Glc-5 all interact with the B/HK HA receptor-binding site. Gal-2 forms a hydrogen bond with the main-chain carbonyl of Leu-237; GlcNAc-3 forms two hydrogen bonds with the side chain of Gln-197, and Glc-5 interacts with Asp-194 and Thr-196. In contrast to LSTc, LSTa exits the receptor-binding site from the N terminus of 190-helix (Fig. 3a).

Fig. 3.

Avian receptor complexes. (a) The interaction of avian receptor analogs in the receptor-binding site of B/HK HA. The hydrogen-bonding interactions are shown as dashed lines in cyan. (b) Comparison of interactions of B/HK and human H3 HAs with avian receptor analogs. The ≈60° rotation of the GlcNAc-3 ring of the avian receptor analog in the receptor-binding site of B/HK HA is indicated by an arrow.

The bound LSTa in B/HK HA is in a cis-conformation, most similar to that of LSTa in human H3 (Fig. 3b) and swine H9 HAs, and in sharp contrast to the trans-conformation of LSTa in avian H3 (17), avian H5 (15), and human 1934 H1 HAs (16). In the cis-conformation, the hydrophobic side chain of residue Pro-238 and the hydrogen bond between Gal-2 and main-chain carboxyl of Leu-237 play an important role, just as Leu-226 in human H3 and swine H9 HAs (11, 15). In the B/HK HA–LSTa complex, the GlcNAc-3 rotates around the Gal-2–GlcNAc-3 bond by ≈60° in relation to GlaNAc-3 of LSTa in human H3 HA. It is this rotation that directs the rest of LSTa toward the 190-helix and allows for the interaction of Glc-5 with Asp-194 and Thr-196 (Fig. 3b). As a result, LSTa fits more snugly in the receptor-binding site of B/HK HA. In sharp contrast, LSTa in human H3 HA moves away from the 190-helix at as early as GlcNAc-3 (Fig. 3b).

The significant contributions of the asialo portion of avian receptor to the interaction with influenza B virus HA are consistent with its substantially higher affinity for larger receptors than for Neu5Ac (41). In sharp contrast, the H1 and H3 subtype HAs have comparable affinity for larger receptors to that of free Neu5Ac (41), which agrees well with the less extensive interactions made by the asialo portion of LSTa with those HAs (11, 14, 16).

Discussion

One unique feature of the B/HK HA receptor-binding site is the presence of Phe-95 in place of tyrosine as ordinarily found in all known influenza A virus HAs. It is interesting to note that a Tyr-98→Phe mutation of human H3 HA abolished erythrocyte binding, and the mutant virus could not infect mutant MDCK cells with a reduced level of cell-surface sialic acid, underscoring the importance of the hydrogen bonds that Tyr-98 makes with His-183 and with receptors inside the receptor-binding site of H3 HA (24). However, in the case of influenza B/HK HA, although these hydrogen bonds are absent, the capability of the virus to bind to cell-surface receptors is not abolished. Given the close evolutionary relationship between influenza A and B virus HAs, it remains an open question as to whether Phe-95 in influenza B virus HA is a direct result of selective pressure to optimize the receptor-binding site or it is simply a random mutation that has survived in evolution. In other words, it needs to be determined whether the receptor-binding site of influenza B virus HA is still able to accommodate the extra hydroxyl group of a tyrosine residue at position 95. Further investigation is needed to help address this question.

Naturally influenza B virus has been detected only in humans. When adapted to growth in chicken eggs, changes in the properties of influenza B virus had been noticed (51). Egg adaptation frequently caused the loss of a glycosylation site at residues 194 to 196 (42) and affects both antigenicity (52–54) and receptor-binding properties of HA (40, 53). The egg-adapted influenza B virus variants, with the absence of the glycans at residue 194, tend to grow better in chicken eggs than mammalian cell propagated ones of the same origin (54). The B/HK HA–LSTa complex structure provides a highly plausible explanation for why the loss of the glycosylation site at residues 194 to 196, as in the case of influenza B/HK HA, is advantageous for egg-adapted influenza B virus (54). It is because the glycans attached to Asn-194 in wild-type influenza B virus HA severely interfere with the binding of α(2,3)-linked sialic acids that are abundant in chicken egg allantone (40), thereby hindering the propagation of influenza B virus. Thus, the growth environment of chicken eggs imposes a strong selective pressure on influenza B virus toward losing the glycans at residue 194, with concomitant enhanced affinity for α(2,3)-linked sialic acids (40). Interestingly, the gain or loss of a glycosylation site at residue 194 seems to be an effective tool used by naturally occurring influenza B virus to modify its antigenicity (55, 56). Similar strategy was also found by egg-adapted influenza A virus (2, 40).

Superposition of all known structures of HA complexed with human receptor analog LSTc on the Sia-1 moiety reveals two different conformations of the bound analog (Fig. 2d): a slightly more extended conformation observed in 1930 H1 and 1934 H1 HAs and a more folded conformation as seen in avian H3, human H3, swine H9, and B/HK HAs. In the more extended conformation, the Gal-2 moiety inserts deeply into the receptor-binding site and interacts with the main-chain carbonyl of residue 225 as well as with the side chain of residue Lys-222. In sharp contrast, the Gal-2 moiety in the more folded conformation is positioned by ≈1–2 Å higher in the binding site (16). To understand the structural basis for the observed conformational differences of LSTc, we compared the residues at position 226 and 222 that interact with LSTc in the complex structures (Table 2). Residue 226 has been implicated in determining the preferential binding of H3 HA with α(2,6)-linked or α(2,3)-linked receptor analogs: Leu-226 prefers α(2,6)-linked receptors, whereas Gln-226 prefers α(2,3)-linked receptors (2, 20, 21). However, despite the difference at residue 226, the α(2,6)-linked receptor LSTc binds to both avian and human H3 HAs in a folded conformation, pointing to an important role of Trp-222 that is common for both H3 HAs (Table 2). In agreement with this finding, swine 1930 H1 and human 1934 H1 HAs, both having the same residue as avian H3 HA at 226 (Gln-226) but different residues at 222 (Lys-222 for both H1 HAs), bind to human receptor analogs in a different, more extended conformation. Thus, it appears that residue 222 is likely a major determinant for the conformations of bound α(2,6)-linked receptor analogs. In particular, a hydrophilic residue such as lysine at 222 offers favorable hydrogen bonding interaction with Gal-2 to pull it to a lower position. In contrast, a hydrophobic residue at position 222, tryptophan in avian and human H3 HAs, or leucine in swine H9 and human B/HK HAs, tends to push Gal-2 up to a higher position, possibly to avoid unfavorable interactions between hydrophilic groups on Gal-2 and hydrophobic groups on residue 222 (Fig. 2d). In the case of B/HK HA, a weak hydrogen bond between the main-chain carbonyl of Leu-237 and Gal-2 (Fig. 2a) brings Gal-2 to a slightly lower position than its equivalence in human H3 and swine H9 HAs.

Table 2.

Comparison of residues interacting with human receptor analogs in different HA proteins

Strain	Residue at 226	Residue at 222	Conformation of LSTc
Swine 1930 H1	Gln	Lys	Extended
Human 1934 H1	Gln	Lys	Extended
Avian H3	Gln	Trp	Folded
Human H3	Leu	Trp	Folded
Swine H9	Leu	Leu	Folded
Human B/HK	Pro-238	Leu-237	Folded

Materials and Methods

Influenza B/HK HA protein was digested with bromelain to remove the C-terminal transmembrane sequence of HA2 (57). Pure B/HK HA protein at 15 mg/ml was used for crystallization trials. Crystals of diamond shape were grown in 100 mM Pipes (pH 6.5) and 2.5 M ammonium sulfate at 18°C within a week to 100 × 100 × 50 μm. Crystals were soaked in mother liquor containing 16 mM LSTa or LSTc (Sigma) for 30 min and then transferred to mother liquor containing 9.0% glycerol before they were flash-frozen in liquid nitrogen. The diffraction data were collected at Advanced Photon Source beamline BM14C. All data sets were processed by using the HKL2000 package (58). The structure of unbound B/HK HA was used as a phasing model (Q.W., F. Cheng, M. Lu, X.T., and J.M., unpublished work). Model building was carried out by using the program O (59), and structural refinement was carried out by using CNS (60) and REFMAC5 in CCP4 (61). Ten percent reflections of the native data set were set aside for calculating R_free factor. The quality of the model was analyzed by using PROCHECK in CCP4 (61). Structural alignment of B/HK HA with influenza A virus HA molecules was performed by using LSQKAB in CCP4 (61) superimposed at the 140-loop, 240-loop, 190-helix, and the regions that harbor the key residues His-191 and Trp-158 of the receptor-binding site. Figures were made by using MOLSCRIPT software (62) and rendered by using RASTER3D (63).