Sialosides and sialylglycopolymers.

Free N-acetylneuraminic acid (Neu5Ac), α-methyl glycoside of N-acetylneuraminic acid (MN; Neu5Acα2Me), and 3′-sialyllactose (3′SL; Neu5Acα2-3Galβ1-4Glc) were purchased from Sigma. 6′-Sialyl(N-acetyllactosamine) (6′SLN) was a gift from V. E. Piskarev, Nesmeyanov Institute of Organoelement Compounds, Moscow, Russia. The sialylglycopolymers 3′SL-PAA and 6′SLN-PAA, containing 20 mol% of 3′SL and 6′SLN, respectively, and 5 mol% of biotin attached to a soluble polyacrylamide carrier, were synthesized as described previously (3). The sialidase inhibitor zanamivir (2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-d-neuraminic acid; GG167) was kindly provided by R. Bethell, Glaxo Wellcome R&D.

Assay of virus-binding affinity for sialylglycopolymers.

The general protocol for this solid-phase receptor-binding assay was described previously (12). Modifications of the assay for the present study were limited to the use of synthetic sialylglycopolymers and of a biotin-streptavidin detection system. In brief, 50-μl aliquots of bovine fetuin solution in phosphate-buffered saline (PBS; 5 μg/ml) were incubated in 96-well polyvinyl chloride microplates (Costar) at 4°C overnight. The plates were washed with water and air dried. Concentrated virus stocks were diluted with PBS to an HA titer of 20 to 100. Then, 40 μl of virus solution was added to each well of the fetuin-coated microplates. After incubation at 4°C overnight, the plates were washed with ice-cold washing buffer (WB; 0.01% Tween 80 in 0.2× PBS). Serial twofold dilutions of sialylglycopolymers in the reaction buffer (RB; 0.02% Tween 80, 0.02% bovine serum albumin, 1 μM sialidase inhibitor GG167 in PBS) were added into the wells (20 μl/well), and the plates were incubated at 4°C for 2 h. After washing, streptavidin-peroxidase solution in RB (1/2,000) was added at 25 μl/well, and the plates were incubated at 4°C for 1 h. After washing, the peroxidase activity in the wells was assayed with o-phenylenediamine substrate solution. The absorbancies (at 490 nm) were determined with a model 3550 microplate reader (Bio-Rad), transferred to a PC using Microplate Manager 4.0 software (Bio-Rad), and processed with Microsoft Excel software. The data were converted to Scatchard plots (A₄₉₀/C versus A₄₉₀, where C is the concentration of the sialylglycopolymer and A₄₉₀ is the absorbency in the corresponding well; see Fig. ​Fig.1).1). The apparent association constants of virus complexes with sialylglycopolymers (K_ass) were determined from the slopes of these plots and are expressed in micromolar amounts of Neu5Ac (with respect to concentration of sialic acid residues present in the solution). Because the assay includes extensive washing steps and is based on a complex multivalent binding of sialylglycopolymers to a virus with numerous HAs, these constants are not true equilibrium association constants. However, they provided a reliable and convenient measure of the relative affinity of viruses for the sialylglycopolymers. Thus, although the absolute values of the apparent association constants determined on different days varied up to fourfold, the relative affinities of the viruses were highly reproducible. The data presented in the tables are average values of two to three independent experiments performed on different days.

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FIG. 1

Binding of sialylglycopolymers 3′SL-PAA (solid lines) and 6′SLN-PAA (dotted lines) by H3 subtype influenza virus strains A/duck/Hokkaido/33/80 and A/Los Angeles/2/87. The binding assay is described in Materials and Methods. The upper panels represent the primary data (dependence of absorbancy in the wells, A₄₉₀, versus concentration of the polymer); the lower panels show the corresponding Scatchard plots.

Assay of virus binding of sialic acid and sialosides.

The apparent association constants of the influenza virus complexes with low-molecular-mass receptor analogs were determined in a fetuin-binding inhibition assay, as previously described (12, 22). This assay is based on the competition between the receptor analog under study and a standard preparation of peroxidase-labeled fetuin for the binding sites on a solid-phase immobilized virus. The competitive reaction was performed for 1 h at 2 to 4°C in the presence of 1 μM GG167 to block the activity of viral neuraminidase.

Alterations of the avian H3 HA at the beginning of the 1968 Hong Kong pandemic.

The reassortant virus possessing an avian H3 HA and all other genes from a previously circulating human virus caused the 1968 pandemic. A few mutations in the HA, including those at positions 144, 193, 226, and 228, were found to separate the earliest human isolate Aichi/2/68 from its putative avian virus precursor (2) (see Fig. ​Fig.33 for the location of these mutations on the HA molecule). To determine the contribution of these mutations to the receptor-binding properties, we compared sialylglycopolymer-binding activities of viruses described by Bean et al. (2). All H3 avian viruses exhibited the same binding specificity, that is, they bound 3′SL-PAA more than an order of magnitude better than they did 6′SLN-PAA (Table ​(Table1).1). Three of the avian H3 viruses tested (duck/Hokkaido/8/80, mallard/NY/6874/78, and duck/Hokkaido/7/82) differed from other avian viruses by having arginine or serine instead of glycine in HA position 228 (Fig. ​(Fig.2,2, H3). The first two strains and duck/Ukraine/63 virus reproducibly showed two- to threefold lower affinities for 3′SL-PAA compared to duck/Hokkaido/7/82 and other H3 avian strains. Although duck/Ukraine/63 bears “avian” 226Q and 228G (amino acids conserved among the majority of avian HAs), it also has a nonconservative substitution, 227S→P, in the HA that can affect the structure of the region surrounding this residue. Because dk/Hokkaido/7/82 was reported to contain 228S (16) but did not differ in our experiments from H3 viruses with 228G, the HA sequence of the preparation of the virus we used in the binding studies was determined. The HA was found to contain glycine at position 228. These findings indicated that the presence of an “atypical” amino acid at position 228 (and 227) correlated with a lower affinity for 3′SL-PAA. Mutations 228G→R and 227S→P did not substantially increase the affinity for 6′SLN-PAA.

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FIG. 2

Partial HA amino acid sequences (HA1 positions 130 to 230) of influenza viruses that were tested for their binding to sialylglycopolymers. Full names of the virus strains are listed in Tables ​Tables11 to ​to3.3. Differences with respect to the top sequence are shown. The H3 numbering system is used; the position in the H1 HA that is absent from the H3 HA is indicated by an underscore. The RBS line shows the position of the amino acid with respect to the HA receptor-binding site. The “R” designates that the amino acid residue contacts either sialic acid or penultimate galactose in the X31 virus HA complex with 3′-sialyllactose (1HGG structure; Brookhaven Protein Database). The star indicates that an amino acid is within 15 Å of the C2 carbon atom of the sialic acid in the 1HGG structure. The figure was generated with GeneDoc 2.3 software (25).

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FIG. 3

Positions of amino acids in the HA receptor-binding site that differ between early epidemic human and swine viruses and their closely related avian counterparts (stereo view). The figure is based on the crystallographic model of the X31 HA complex with the Neu5Acα2-6Gal-containing receptor analog LSTc (Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc) (10). The solvent-accessible molecular surface of the protein is shown. The sialic acid residue and penultimate galactose ring of LSTc are displayed as thick stick bonds; the rest of the molecule is shown as a thin white line. The gray transparent sphere (C6) in close proximity to amino acid 226 represents the van der Waals surface of the C6′-carbon atom of Gal. The figure was generated with WebLab ViewerPro 3.10 (Molecular Simulations, Inc., San Diego, Calif.).

All human H3 isolates, including the earliest, Aichi/2/68, bound to 6′SLN-PAA with a higher affinity and to 3′SL-PAA with a lower affinity than did all of the avian viruses tested. The Aichi/2/68 HA bears substitutions 144G, 182I, 193S, 226L, and 228S with respect to the avian HA consensus sequence (Fig. ​(Fig.2).2). The 182V→I mutation of Aichi/2/68 is unique and is absent in other human virus strains; hence, it is unlikely to be critical for the “human” receptor-binding phenotype. Because 144G is present in the HA of the avian virus mallard/NY/6874/78 that does not bind to 6′SLN-PAA any better than other avian viruses, it is probably not essential for the increased affinity of human viruses for 6′SLN-PAA. The duck/Memphis/74 strain reproducibly bound to 6′SLN-PAA more weakly than any other avian virus. This feature correlates with the presence of 193D in this HA receptor-binding site (RBS), whereas other avian viruses bear 193N, and the early human strains bear 193S. Residue 193 is relatively far from the region of the RBS, which accommodates the Neu5Acα2-6Gal moiety (10), but it can potentially interact with more distant saccharide rings of the Neu5Acα2-6Gal-terminated receptors and with the polymeric part of 6′SLN-PAA (Fig. ​(Fig.3).3). Although these findings suggest that mutations at position 193 can modulate the recognition of 6′SLN-containing receptors, substitutions in positions 226 and 228 appear to be primarily responsible for the differences in the receptor specificity between avian viruses and Aichi/2/68.

To further evaluate the effects of mutations 226Q→L and 228G→S on changes of the receptor-binding properties of avian HA, we analyzed laboratory variants of the human virus Udorn/307/72, which possessed distinct amino acids at these positions (Table ​(Table1).1). The R2 variant, for example, bears “avian” amino acids at both positions and is indistinguishable from the natural avian H3 viruses with respect to its binding to sialylglycopolymers. Thus, in the presence of 226Q/228G, other amino acids that differ from avian viruses (i.e., at positions 144, 155, 188, and 193) do not appear to substantially affect the avian virus-like receptor-binding specificity of the R2 virus. The R4 variant has a single mutation, 228G→S, with respect to R2. This substitution has no effect on the affinity of the HA for 6′SLN-PAA but reduces its affinity for 3′SL-PAA, similar to the effect of substitution 228G→R in the avian viruses described above. By contrast, the substitution 226Q→L (compare the R4 variant with its parent, Udorn/72) markedly changes the receptor-binding properties of the virus by both substantially increasing its affinity for 6′SLN-PAA and decreasing its affinity for 3′SL-PAA. These data show that the 226Q→L mutation primarily determines the change in recognition of the Neu5Ac-Gal linkage during interspecies transfer, whereas substitutions of glycine at position 228 of the avian HA have only limited effect on the Neu5Ac-Gal linkage specificity.

Alterations of the avian H2 HA at the beginning of the Asian pandemic.

The “Asian” pandemic of 1957 was caused by a reassortant virus that contained the genes of the surface glycoproteins and the PB1 protein from an H2N2 avian strain and the remainder from a human virus. We analyzed the receptor-binding properties (Table ​(Table2)2) and amino acid sequences (Fig. ​(Fig.2,2, H2) of the panel of H2 avian viruses (35) and the earliest known H2 human strains (17).

TABLE 2

Binding of sialylglycopolymers to H2 influenzaviruses

Virus strain	Amino acid at position:		Abbreviation	Apparent association constant (Kass, μM−1 Neu5Ac)a		Relative affinityb
	226	228		3′SL-PAA	6′SLN-PAA
Avian
Mallard/Potsdam/178/83	Q	G	maPot/83	90	1.5	60
Peking dk/Potsdam/1689/85	Q	G	pdkPot/86	65	1	65
Pintail/Praimorie/625/76	Q	G	pinPraim/76	100	1	100
Duck/Hong Kong/273/78	Q	G	dkHK/78	50	1	50
Mallard/MT/61	Q	G	maMT/61	100	2	50
Herring gull/DE/677/88	Q	G	hguDE/88	110	2	55
Gull/MD/19/77	Q	G	guMD/77	40	1	40
Mallard/ALB/353/88	Q	G	maALB/88	95	1	95
Guinea Fowl/NJ/3070/91	Q	G	gfNJ/91	70	1	70
Mallard/ONT/56/76	Q	G	maONT/76	40	1.2	33
Mallard/NY/6750/78	Q	G	maNY/78	60	2	30
Human
El Salvador/2/57	Q	G	Sal/57	50	1	50
Leningrad/134/57	Q	G	Len/57	50	1	50
RI/5−/57	Q	G	RI5−/57	40	0.5	80
Davis/1/57	L	G	Dav/57	1.5	12	0.13
Albany/7/57	L	G	Alb/57	1	12	0.08
RI/5+/57	L	S	RI5+/57	1	25	0.04
Albany/6/58	L	S	Alb/58	1	30	0.03
Malaya/16/58	L	S	Mal/58	1	25	0.04
Sao Paolo/3/59	L	S	SP3/59	1	25	0.04
Victoria/15681/59	L	S	Vic/59	1.5	20	0.075
Ohio/2/59	L	S	Ohio/59	2	25	0.08
Ann Arbor/6/60	L	S	AA6/60	25	2.5	10
Berlin/3/64	L	S	Ber/64	5	30	0.17

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^aThe binding assay was performed as described in Table ​Table1,1, footnote a. 

^bSee Table ​Table1,1, footnote b. 

All H2 avian viruses contained 226Q and 228G and bound strongly to 3′SL-PAA and weakly to 6′SLN-PAA. Among the H2N2 viruses isolated from humans in the first year of the pandemic, three strains (El Salvador/57, Leningrad/57, and RI/5−/57) carried the amino acids typical for avian viruses, 226Q and 228G, and displayed the avian virus-like receptor-binding phenotype. In contrast to these strains, Davis/57 contains only one amino acid substitution in the region of the receptor-binding site, 226Q→L, whereas Albany/57 contains two substitutions, 226Q→L and 186N→I. Both of these variants with 226L displayed a dramatic shift in receptor-binding activity, with their affinity for 6′SLN-PAA increasing ca. 10-fold and that for 3′SL-PAA decreasing >10-fold. Thus, these two strains are similar to the earliest human H3 pandemic strain, Aichi/2/68, with respect to their affinity for sialylglycopolymers. The RI/5+/57 strain and other H2 human viruses isolated in later years displayed a further twofold increase in affinity for 6′SLN-PAA; this feature correlates with the presence of serine at position 228 of the HA. Among the human H2 viruses, the Ann Arbor/6/60 strain showed the most unusual receptor-binding properties. Despite the presence of 226L/228S, the receptor-binding specificity of this strain is closer to that of the avian viruses. Three substitutions in the RBS region (137R→Q, 158G→E, and 186N→I; Fig. ​Fig.2,2, H2), which separate this strain from the human consensus sequence, could be responsible for the altered binding specificity. Mutations in HA positions 137 and 186 were previously found to be involved in the adaptation of human H3N2 virus to growth in the presence of animal sera, the receptor specificity of the serum-resistant variants being similar to that of Ann Arbor/60 (20).

Receptor-binding specificity of H3 viruses from seals.

In 1991, H3N3 avian virus-like viruses were isolated from lung tissues of seals that died of pneumonia along the Cape Cod peninsula of Massachusetts (5). We examined four seal viruses isolated at different times during the outbreak (Table ​(Table1);1); the HA sequences of two had been determined earlier (Fig. ​(Fig.2,2, H3). Three of the four isolates displayed a typical avian virus-like pattern of binding to sialylglycopolymers, that is, an affinity for 3′SL-PAA of about 50 μM⁻¹ and an affinity for 6′SLN-PAA of about 1 μM⁻¹. This finding suggests that an avian influenza virus can infect seals without substantial changes in its receptor-binding specificity. By comparison with three other H3 seal viruses, seal/MA/3984/92 showed a lower binding affinity for 3′SL-PAA with no increase in binding to 6′SLN-PAA. The reasons for this discrepancy are not presently clear. Two amino acid substitutions in the upper portion of the HA globular head, 138A→S and 220R→S, separate this strain from the seal/MA/3911/92 virus. Both substitutions involve amino acid residues that are conserved among influenza virus HAs of all antigenic subtypes (23, 26). Position 220 is located on the boundary between the HA monomers relatively distant from the RBS. Amino acid 138 resides in the receptor-binding pocket (Fig. ​(Fig.3)3) and is much more likely to be responsible for the decreased binding to 3′SL-PAA.

Alterations of avian H1N1 viruses during their replication in pigs.

In 1979, an avian virus-like virus was isolated from pigs (36). This virus continues to circulate in European pigs (4, 18) and provides a rare opportunity for investigation of the receptor-binding specificity changes during adaptation of an avian virus to a new host. Besides so-called avian-like swine viruses, “classical” H1N1 swine influenza virus strains were included in this study to represent the receptor-binding properties of viruses that have circulated in pigs for about 80 years and are highly adapted to this host (Fig. ​(Fig.44 shows phylogenetic relationships among H1 avian, swine, and human viruses).

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FIG. 4

Phylogenetic tree for H1 subtype influenza viruses from different hosts, as determined based on the amino acid sequences of the HA1. The tree was constructed as described in Materials and Methods using the HA sequences of viruses listed in Fig. ​Fig.22 and the following sequences of classical swine and human viruses from GenBank: sw/Iowa/15/30, WSN/33, PR/8/34 (Cambridge strain), FM/1/47, USSR/90/77, Taiwan/1/86, and Finland/168/91. The figure was generated with the TREEVIEW 1.5.2 program (27).

We found that the receptor-binding characteristics of H1N1 avian strains are similar to those of H2 and H3 avian viruses (Table ​(Table3).3). By contrast, the early avian-like swine isolates swine/Arnsberg/79 and swine/Netherlands/80 showed a substantial increase in affinity for 6′SLN-PAA. Notably, these swine viruses bound 6′SLN-PAA with the same affinity as do the early human pandemic strains Albany/57 (H2N2), Davis/57 (H2N2), and Aichi/68 (H3N2). Unlike H2 and H3 human viruses, the early avian-like swine viruses retained the ability of their avian predecessors to bind 3′SL-PAA. However, the affinity for 3′SL-PAA shown by swine isolates from subsequent years was gradually reduced, reflecting continued evolution of the virus in swine. The three more recent avian-like swine viruses isolated in 1987, 1991, and 1992 displayed binding affinities that were similar to those of classical swine viruses.

Four amino acid substitutions affecting the avian virus consensus sequence are present in the region of the receptor-binding site of the early avian-like swine viruses swine/Arnsberg/79 and swine/Netherlands/80 (positions 155, 159, 190, and 225; see Fig. ​Fig.2,2, H1, and Fig. ​Fig.3).3). One or more of these substitutions should be thus responsible for the increase in the affinity of avian H1 HA for the 6′SLN-PAA-containing glycopolymer. We therefore compared amino acid residues at these positions among the H1 HAs of classical swine and human viruses (Table ​(Table4).4). These positions were conserved in all 13 H1 avian HAs analyzed. Although classical swine viruses and avian-like swine viruses belong to distinct phylogenetic lineages (Fig. ​(Fig.3),3), most strains of both lineages carry the same substitutions, 155T→V, 159T→N, and 190E→D, suggesting that these changes are essential for adaptation of the avian HA to swine hosts. The mutation 225G→E is present in the HA of two early avian-like swine viruses but is absent in later isolates (Fig. ​(Fig.2)2) and in the classical swine strains. Thus, although this substitution could be involved in the early stages of adaptation of the avian H1 HA to swine, it was not preserved during subsequent replication of the virus in this host.

TABLE 4

Amino acids at positions 155, 159, 190, and 225 of the HA of H1 viruses from birds, swine, andhumans^a

Virus type (no. of sequences analyzed)	Amino acid residue at position:
	155	159	190	225
H1
Avian (13)	T*	T*	E*	G*
Classical swine (22)	V*	N[19], D[2], S[1]	D[21], E[1]	G[20], D[1], N[1]
Avian-like swine (8)	V[6], I[2]	N*	D[7], E[1]	G[6], E[2]
Human (42)	T*	G*	D[33], E[5], N[4]	D[22], G[19], N[1]
Human, 1918 (3)b	T*	S*	D*	D[2], G[1]
H2-H15 avian (14)c	Variable: T, V, I, L	Variable: G, A, S, T, N, Q, D	E*	G[13], N[1]

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^aThe analysis was performed with sequences obtained from the GenBank and from the literature (29, 41). An asterisk next to an amino acid indicates that it was conserved in all sequences. Otherwise, the number of sequences for a particular amino acid is given in brackets. 

^bReid et al. (30). 

^cThe analysis was performed with a previously described set of avian virus HA sequences representing each of the 15 HA subtypes (23). 

Among the human viruses, no changes in the avian sequence were detected at position 155, whereas a substitution at position 159 (T→G) differed from that in swine viruses (T→N or T→S). Because, similar to swine viruses, H1N1 human viruses display a high binding affinity for 6′SLN-PAA (13), one can conclude that amino acids at these two positions are not of primary importance for recognition of the 6′SLN determinants. What, then, can be the role of these substitutions for HA adaptation to swine? The side chain of the amino acid at position 155 participates in the formation of the pocket that accommodates the acyl substituent at 5-N of Neu5Ac (see Fig. ​Fig.3).3). Thus, we hypothesize that the substitution 155T→I/V in the swine virus HA increases the affinity of the virus for 5N-glycolyl analog of the sialic acid that is abundant in pigs but absent in birds and humans (see reference 38 and references therein). Amino acid 159 is at the tip of the HA (Fig. ​(Fig.3)3) and therefore could potentially affect interactions of viruses with the distant parts of the sialyloligosaccharides or with protein parts of the receptors.

Most human viruses bear the same substitution as swine viruses at position 190 (E→D) and carry a similar substitution at position 225 (G→D). In fact, every swine and human H1 HA analyzed in this study differed from the avian consensus at least in one of these positions (Table ​(Table4).4). Thus, mutations at positions 190 and 225 correlate most closely with the ability of the virus to bind 6′SLN and appear then to be primarily involved in the adaptation of the H1 avian virus HA to swine and humans.

We thank Peng Gao for sequencing the HA1 gene of dk/Hokkaido/7/82 virus, V. E. Piskarev for the generous gift of 6′SLN, and M. B. Eisen for crystallographic structures of influenza virus HA complexes with sialyloligosaccharides. We are grateful to R. Bethell of GlaxoWellcome R&D for providing the NA inhibitor zanamivir (GG167). We also thank Krisna Wells and Martha McGregor for technical assistance and J. Gilbert for editing the manuscript.

This work was supported by Public Health Service research grants from the National Institute of Allergy and Infectious Diseases, by Cancer Center Support (CORE) grant CA-21765, and by the American Lebanese Syrian Associated Charities. Mikhail Matrosovich was supported by a Karnofsky fellowship from St. Jude Children's Research Hospital.