M. P. Chumakov Institute of
Poliomyelitis and Viral Encephalitides, 142 782 Moscow,
Russia1;
Department of Virology and
Molecular Biology, St. Jude Children's Research Hospital, Memphis,
Tennessee 381052;
Department of
Pathobiological Sciences, School of Veterinary Medicine, University
of Wisconsin
Madison, Madison, Wisconsin
537063; and
Department of Pathology,
University of Tennessee, Memphis, Memphis, Tennessee
381634
H3N2 human influenza viruses that are resistant to horse, pig, or
rabbit serum possess unique amino acid mutations in their hemagglutinin
(HA) protein. To determine the molecular mechanisms of this resistance,
we characterized the receptor-binding properties of these mutants by
measuring their affinity for total serum protein inhibitors
and for soluble receptor analogs. Pig serum-resistant variants
displayed a markedly decreased affinity for total pig serum
sialylglycoproteins (which contain predominantly 2-6 linkage between
sialic acid and galactose residues) and for the sialyloligosaccharide 6'-sialyl(N-acetyllactosamine). These properties
correlated with the substitution 186S
I in HA1. The major
inhibitory activity in rabbit serum was found to be a 
inhibitor
with characteristics of mannose-binding lectins. Rabbit serum-resistant
variants exhibited decreased sensitivity to this inhibitor due to the
loss of a glycosylation sequon at positions 246 to 248 of the HA. In
addition to a somewhat reduced affinity for
6'-sialyl(N-acetyllactosamine)-containing receptors,
horse serum-resistant variants lost the ability to bind
the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties
of equine 
2-macroglobulin because of the mutation
145NK/D in their HA1. These results indicate that influenza viruses
become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we
compared the receptor-binding properties of H3N8 avian and equine
viruses, including two strains isolated during the 1989 to 1990 equine
influenza outbreak, which was caused by an avian virus in China. Avian
strains bound 4-O-acetylated sialic acid residues of equine
2-macroglobulin, whereas equine strains did not.
The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian
viruses; a later isolate, however, displayed binding properties more
similar to those of classical equine strains. These data suggest that
the neuraminidase-resistant sialylglycoconjugates present in horses
exert selective pressure on the receptor-binding properties of avian
virus HA after its introduction into this host.
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INTRODUCTION |
Influenza A viruses possess
two envelope glycoproteins:hemagglutinin (HA) and neuraminidase (NA).
HA binds to cell surface sialylglycoconjugates and mediates virus
attachment to target cells (19, 30). NA cleaves the
-glycosidic linkage between sialic acid and an adjacent sugar
residue, facilitating elution of virus progeny from infected cells and
preventing self-aggregation of the virus (1, 13). Natural
sialylglycoconjugates are structurally diverse (37, 40), and
the preferential recognition of distinct sialyloligosaccharides by HA
and NA correlates with the host species from which the viruses are
isolated (reviewed in references 19, 30, and
38; see also references 4, 6, 7,
11, and 28).
The receptor-binding activity of influenza viruses can be inhibited by
certain molecules present in the sera and fluid secretions of animals
(see references 14 and 21 for
reviews). These inhibitors are classified as , , and 
types
based on their thermal stability, virus-neutralizing activity, and
sensitivity to inactivation by NA and periodate treatments. The inhibitors are thermolabile mannose-binding lectins that interact with
the oligosaccharide moieties on viral glycoproteins. They
neutralize virus by steric hindrance of HA and by activation of the
complement-dependent pathway (2, 3). By contrast, the and inhibitors are heat-stable sialylated glycoproteins
that mimic the structure of the cellular receptors of influenza viruses
and competitively block the receptor-binding sites of HA. Influenza
viruses are neutralized by inhibitors but not by inhibitors,
which are considered to be sensitive to viral NA. However, the
distinction between and inhibitors is strain dependent and
rather arbitrary, as described by Gottschalk et al.
(14). Although inhibitors in serum or other body fluids are
believed to influence the selection of influenza virus receptor
variants in natural hosts, no direct experimental support for this
hypothesis has been presented.
A potent inhibitor of H2 and H3 human influenza viruses, equine
2-macroglobulin (EM), contains a
Neu4,5Ac22-6Gal moiety that is insensitive to viral NA and
thus resists inactivation by this enzyme (16, 24, 31).
Cultivation of human H3 influenza viruses in the presence of
horse serum results in the selection of variants that have a
decreased affinity for the Neu5Ac2-6Gal-specific receptors due to a
single amino acid substitution (226LQ) in their HA (32,
33). One of these mutants (X31/HS strain) does not bind the
Neu4,5Ac2 (4-O-acetylated sialic acid) species
(25). Therefore, there are at least two mechanisms by which
a virus can become resistant to the horse serum inhibitor: a change in the recognition of the type of Sia-Gal linkage, and a change in the
recognition of the 4-O-acetylated sialic acid. The relative contributions of these mechanisms to the resistant phenotype are yet to
be defined.
We have previously shown that horse, pig, and rabbit sera all contain
distinct heat-resistant inhibitors of the H3N2 human influenza virus
A/Los Angeles/2/87 (LA/87), because variants resistant to these sera
possess unique mutations in their HA receptor-binding regions
(34). The major inhibitor in pig serum was later identified as 2-macroglobulin that contains predominantly 2-6 linkage between sialic acid and galactose (35). Gimsa et al.
(12) recently showed that pig serum-resistant
human and swine strains exhibit decreased affinity for human
erythrocytes that had been modified to contain terminal
Neu5Ac2-6Gal residues. However, the nature of the rabbit
serum inhibitor and the mechanisms of influenza virus resistance to
each serum inhibitor remain unknown.
To understand the molecular mechanisms by which influenza viruses
become resistant to horse, pig, and rabbit serum inhibitors, we
compared the receptor-binding characteristics of LA/87 and its
serum-resistant variants and analyzed these data in relation to the
known amino acid substitutions in the HA of the mutants. We then
analyzed the receptor-binding properties of viruses isolated during
an equine influenza outbreak that was caused by an avian virus, in
order to evaluate the influence of natural inhibitors on the evolution
of virus in a new host.
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MATERIALS AND METHODS |
Viruses.
Variants were isolated from the parent virus
LA/87 by growth in embryonated chicken eggs in the presence of horse,
pig, or rabbit serum, or their mixtures, and characterized as
previously described (24) (Table
1). Avian and equine influenza A virus strains were from the virus repository of St. Jude Children's Research
Hospital; their isolation and characteristics have been described
elsewhere (5, 15). All viruses were grown in 9- to
10-day-old chicken eggs. The allantoic fluids were clarified by
low-speed centrifugation; the viruses were pelleted by high-speed centrifugation, resuspended in 0.1 M Tris buffer (pH 7.2) containing 50% glycerol, and then stored at 
20°C.
Soluble receptor analogs and serum inhibitors.
Sera were
obtained from Pel-Freez Biologicals (Rogers, Ark.) and stored at
20°C in aliquots. Free N-acetylneuraminic acid (Neu5Ac), 3' sialyllactose (3' SL; Neu5Ac2-3Gal1-4Glc), and all
neutral monosaccharides used were purchased from Sigma, St. Louis, Mo.
6'-Sialyl(N-acetyllactosamine) (6' SLN;
Neu5Ac1-6Gal1-4GlcNAc) was a gift from V. E. Piskarev,
Nesmeyanov Institute of Organoelement Compounds, Moscow, Russia.
Sialylglycopolymers 3' SL-PAA and 6'SLN-PAA, which contained 20 mol%
3'SL and 6'SLN, respectively, attached to a soluble polyacrylamide
carrier were described previously (11). They were kindly
provided by N. V. Bovin and A. B. Tuzikov, Shemyakin
Institute of Bio-Organic Chemistry, Moscow, Russia.
To study heat-stable (sialylglycoprotein) serum inhibitors,
sera were heat inactivated for 30 min at 56°C. To further
discriminate between NA-sensitive and NA-resistant
sialylglycoprotein components, the sera were either treated
with LA/87 virus NA or mock treated. To 60 µl of heat-inactivated
sera, 540 µl of Ca-Tris-buffered saline (Ca-TBS) buffer (6.8 mM
CaCl2, 0.02 mM Tris [pH 7.3], 0.9% NaCl) and 15 µl of
purified LA/87 virus (HA titer of 16,000) were added. The mixture was
then incubated for 4 h at 37°C. Viral HA and NA were inactivated
by heating the mixtures for 1 h at 56°C. This treatment was
determined, through the control experiments, to be sufficient to
eliminate any contribution of the virus to the subsequent assay of
serum inhibitors in the preparations. The mock-treated sera were
handled similarly except that Ca-TBS was used instead of virus.
To study the binding of EM by avian and equine influenza viruses (Table
2), partially purified EM was prepared
from the heat-inactivated horse serum by using gel chromatography as
previously described (29). A fraction of this preparation
(50 µl) in Ca-TBS buffer was incubated with 2.5 mU of Vibrio
cholerae NA (type III; Sigma) at 37°C for 4 h and then at
56°C for 1 h to inactivate the NA. The control EM was treated in
the same way but without the NA incubation.
Rabbit serum inhibitor.
Freshly prepared samples of
rabbit serum inhibitor were used each day to avoid deterioration of
the preparations during storage. The serum was thawed, incubated at
56°C for 30 min, and then chilled on ice. To inactivate sialic acid
containing and inhibitors, 3 ml of 0.011 M NaIO4
was added to 1 ml of serum. After a 15-min incubation at room
temperature, excessive periodate was inactivated by the addition of 0.2 ml of 10% glycerol solution in 1 M Tris buffer (pH 7.3).
Preparation of plasma membranes from CAMs cells.
Chorioallantoic membranes (CAMs) from 12-day-old embryonated chicken
eggs were processed in a Dounce homogenizer with a 0.5-mm clearance to
detach epithelial cells. The cells were pelleted at 2,000 × g and resuspended in 50% Percoll solution in TSE buffer (10 mM Tris, 0.15 M NaCl, 0.5 mM EDTA [pH 7.2]). After centrifugation for
5 min at 10,000 × g, the layer of epithelial cells at
the top of the Percoll solution was removed, and the bottom fractions containing erythrocytes were discarded. This procedure was repeated until no admixtures of erythrocytes were visible. The CAM cells were
then washed from the Percoll in TSE, suspended in an ice-cold lysis
buffer (0.01 M Tris [pH 7.2], 1 mM phenylmethylsulfonyl fluoride),
incubated for 10 min on ice, and disrupted in a standard Dounce
homogenizer. Nuclei and cellular debris were removed by centrifugation
for 1 min at 1,000 × g. The cell membranes were then
pelleted at 40,000 × g for 1.5 h, resuspended in
TSE containing 1 mM phenylmethylsulfonyl fluoride, sonicated for 2 min
in an ice bath, and then stored as aliquots at 20°C.
Virus receptor-binding affinity.
The virus-binding affinity
for serum sialylglycoproteins inhibitors, sialosides, and
sialylglycopolymers was assessed by using the solid-phase
fetuin-binding inhibition assay as previously described (10,
26). This assay is based on the competition for binding sites on
the viral particle between nonlabeled sialic acid-containing compound
and enzyme-labeled sialylglycoprotein fetuin. In brief,
purified viruses diluted with phosphate-buffered saline (PBS) to a
hemagglutination titer of 1:20 to 1:100 were adsorbed to the wells of
fetuin-coated polyvinyl chloride enzyme immunoassay microplates at
4°C overnight. After unbound virus was washed off with 0.01% Tween
80 in PBS (PBS-T), 0.05 ml of solutions containing a fixed amount of
fetuin labeled with horseradish peroxidase (HRP) and a variable amount
of nonlabeled inhibitor were added to the plate, which was then
incubated for 1 h at 4°C. The solutions were prepared in PBS
supplemented with 0.02% bovine serum albumin, 0.02% Tween 80, and 1 µM sialidase inhibitor
2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid (GG167), kindly provided by R. Bethel, Glaxo Wellcome Co. After
this incubation, the plates were washed with PBS-T, and the amount of
labeled fetuin bound was determined by using the standard
o-phenylenediamine chromogenic substrate.
The association constants of the virus complexes with serum
sialylglycoproteins, sialosides, and sialylglycopolymers
were calculated for each concentrations of the compound used in the competitive reaction, and the results were averaged. The association constants for the serum inhibitors were calculated by assigning the
concentration of the inhibitors in each undiluted serum sample a value
of 1 U. Standard deviations of the mean values of the constants
obtained in the same experiment varied from 10 to 40%. The variation
among the mean values of the constants determined in replicate
experiments on different days was higher; however, the overall patterns
of binding affinities of different virus strains for different receptor
molecules were reproducible.
The effect of bivalent ions on the interaction of the virus with the
rabbit inhibitor was studied by using the same assay, with the
following modifications. The virus was incubated separately with the
inhibitor and with the labeled fetuin. First, the solid-phase immobilized virus was allowed to interact with dilutions of the heat-inactivated and periodate-treated serum in various buffers, at
which time the inhibitor attaches to the virions. After washing of
unbound material, fetuin-HRP conjugate in Ca-TBS-bovine serum albumin
buffer, supplemented with 0.05% Tween 80 and 1 µM GG167, was added
for 30 min at 4°C. This step was followed by standard detection of
the bound conjugate (10). The percent inhibition of
conjugate binding to the virus as a result of prebound inhibitor was calculated as 100 × (K+ Ai)/(K+ K
), where Ai is the
absorbancy in the experimental wells, K is the
absorbancy in the control wells without the virus (background conjugate
binding), and K+ is the absorbancy in the
control wells without inhibitor (100% conjugate binding).
Binding of LA/87 and its serum-resistant variants to CAM cell
membranes pretreated with NA.
CAM cell membranes, suspended in PBS
to a final concentration of about 10 µg/ml of total protein, were
incubated in 96-well polyvinyl chloride microplates for 4 h at
4°C. Nonadsorbed material was then washed off with PBS. Wells of the
same microplates that lacked coating were used as background controls.
Adsorbed membranes were treated with decreasing concentrations of
V. cholerae NA (Sigma type III) for 2 h at 37°C or
mock treated (no NA). The viruses were allowed to bind to the
membranes, and the amounts of the viruses bound were estimated by
overlaying them with fetuin-HRP conjugate as previously described
(27). The results were expressed as percent binding to the
NA-treated membranes relative to that to the mock-treated preparation.
Nucleic acid sequencing.
Viral RNA was isolated from
virus-containing allantoic fluid and sequenced as previously described
(20). In brief, cDNA was synthesized with reverse
transcriptase and a random hexamer. The HA genes were amplified by PCR
with the cDNA, H3 HA-specific oligonucleotide primers, and
Pfu polymerase (Stratagene). PCR products were cloned into a
plasmid and sequenced with an Autosequencer (Applied Biosystems Inc.,
Foster City, Calif.) according to the protocol recommended by the
company. Three independent cDNA clones were sequenced and found to be
identical to each other.
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RESULTS AND DISCUSSION |
H3N2 viruses become resistant to horse serum due to their reduced
affinity for 4-O-acetylated sialylglycoconjugates.
The
serum-resistant variants of LA/87 that can grow in embryonated hen eggs
in the presence of heat-inactivated horse, pig, or rabbit serum differ
from their parent virus and from each other in that they possess
distinct HA mutations (34) (Table 1). The goal of this study
is to understand the molecular basis for viral resistance to each
serum. To determine whether those viruses became resistant to serum
because of a reduced binding to NA-sensitive ( inhibitors) and/or
NA-resistant ( inhibitors) sialylglycoconjugates, we compared the
binding affinities of the viruses for serum inhibitors that were either
untreated or treated with the NA of the parent LA/87 virus (Fig.
1). We found that pig and rabbit sera
contained low to undetectable levels of sialylglycoconjugates resistant to the LA/87 NA, because the binding of sialylglycoproteins
in these sera to LA/87 decreased 30- and 45-fold, respectively, after NA treatment (Fig. 1C). Given that we did not optimize the treatment conditions, the residual inhibitory activity in the sera may have been
the result of incomplete treatment rather than the presence of
NA-resistant components.
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FIG. 1.
Binding of inhibitors from animal sera by the LA/87
strain and its serum-resistant variants. Horse (closed bars), pig (open
bars), and rabbit (dashed bars) sera were heat inactivated and either
treated with LA/87 NA or mock treated as described in Materials and
Methods. Association constants of the virus complexes with inhibitors
of mock-treated (A) or NA-treated (B) sera (KMT
and KNA, respectively) were determined in a
competitive solid-phase assay; 5 mM EDTA was present in the assay
buffer to inactivate possible residual -inhibitor activity in the
sera (references 2 and 3 and this
paper). The ratio KMT/KNA
(C) reflects a decrease in the affinity of the serum inhibitors for the
virus after NA treatment. The lower this ratio is, the more resistant
is the inhibitor to inactivation by NA.
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In marked contrast to pig and rabbit sera, horse serum retained up to
30% inhibitory activity against the LA/87 virus after NA treatment,
indicating that substantial amounts of NA-resistant inhibitors are
present in horse, but not in pig or rabbit, serum. Previous studies
have shown that 2-macroglobulin is the principal inhibitor in horse serum for H2N2 and H3N2 viruses and that
4-O-acetylated sialic acid, which represents about 30% of the total
sialic acids in EM (16), confers this inhibitor's
resistance to bacterial and viral NA (16, 24, 25, 31). Our
results are consistent with these previous findings in that LA/87 NA is
unable to cleave 4-O-acetylated sialic acid residues from
2-macroglobulin and in that LA/87 HA does bind this type
of sialic acid.
To understand the molecular basis of horse serum resistance, we
analyzed the receptor-binding properties of serum-resistant variants of
LA/87 (Fig. 1). The serum-resistant variants could be separated into
two distinct groups based on their recognition of the NA-treated EM
(i.e., their ability to bind NA-resistant 4-O-acetylated sialic acid).
Variants LA/87 P and LA/87 R, which were selected from LA/87 with pig
or rabbit serum, respectively, displayed substantial affinity for viral
NA-treated horse serum (Fig. 1B), indicating their ability to bind
4-O-acetylated sialyloligosaccharides. On the other hand, all of the
horse serum-resistant variants lost this ability, as shown by their
dramatic decrease in affinity for the NA-treated horse serum inhibitors
(Fig. 1C; KMT/KNA [see legend to Fig. 1 for definition] = 24 to 160). Similar results were
obtained with V. cholerae NA-treated horse serum, thereby confirming these data (data not shown).
Influenza viruses that become resistant to horse serum inhibitors are
thought to do so because of a reduced affinity for the Sia2-6Gal moiety
of EM (31-33). We therefore analyzed the receptor-binding specificity of serum-resistant viruses by using a panel of receptor analogs (Fig. 2). We found that the LA/87
H virus, a horse serum-resistant variant without any additional
passages with other sera, displayed about threefold-weaker binding
compared to the parent LA/87 virus with respect to the
Sia2-6Gal-containing receptor analogs 6'SLN and 6'SLN-based synthetic
glycopolymer 6'SLN-PAA (Fig. 2). These results suggest that the LA/87
virus acquires resistance to the horse serum inhibitor through a
combination of two mechanisms: reduced binding to NA-resistant
4-O-acetylated sialic acid species present in the inhibitor and reduced
recognition of 2-6-linked sialyloligosaccharide determinants. However,
the first mechanism plays a more important role in horse serum
resistance, because the LA/87 P variant, which has an even lower
affinity for 6'SLN and 6'SLN-PAA than does LA/87 H, binds
4-O-acetylated sialic acid and is horse serum sensitive. This
conclusion highlights the contribution made by viral NA to virus
resistance to neutralization by receptor analogs. This contribution
should be taken into account when developing inhibitors of virus
receptor binding (see references 23 and 29 and references therein).
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FIG. 2.
Binding of receptor analogs by LA/87 and its
serum-resistant variants. The association constants of the virus
complexes with free Neu5Ac, 3'SL, 6'SLN, and sialylglycopolymers of
3'SL and 6'SLN (3'SL-PAA and 6'SLN-PAA) were determined in a
competitive solid-phase assay as described in Materials and Methods.
The constants are expressed in mM1 for the monovalent
sialosides and in µM1 sialic acid for the
sialylglycopolymers. Our data on the binding of total heat-inactivated
pig serum from Fig. 1 (KMT, mU1)
are also represented (Pig serum) to facilitate the comparison of
binding patterns.
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Comparison of the HA amino acid sequences (Table 1) revealed that
substitutions at position 145 of HA1 (either NK or ND) are the
only changes common among all four horse serum-resistant variants and
separate them from other sensitive strains. Therefore, these
substitutions should be primarily responsible for the resistance. The
three-dimensional structure of X31 (H3N2) virus HA complexed with the
-methyl glycoside of 4-O-acetyl-5N-acetylneuraminic acid (36) shows that the 4-O-acetyl group of sialic acid
contacts the side chain of the amino acid at position 145 (Fig.
3). Clearly, changes in the size (NK)
or charge (NK or ND) of the amino acid at this position could
directly interfere with the fit of the 4-O-acetyl moiety to the
receptor binding site.
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FIG. 3.
Key mutations (black) in the HA of serum-resistant
variants of LA/87 shown on a model depicting the complex between X31
virus HA and the -methyl glycoside of
4-O-acetyl-N-acetylneuraminic acid (1HGI
structure; Brookhaven Protein Databank [36]). The
solvent-accessible surface of the receptor-binding site of the HA
monomer A (white) and the adjacent part of the second monomer C (gray)
are represented. The molecule of Neu4,5Ac22Me is shown as a
stick model (heavy atoms only); the carbon and oxygen atoms of the
4-O-acetyl group and the carbon atom of the
2-O-methyl group are shown as white, gray, and black balls,
respectively. Black numbers indicate the positions of the amino acids
discussed in the text. The figure was generated using the Preview
version of WebLab ViewerPro 3.0, Molecular Simulations, Inc., San
Diego, Calif.
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Pig serum-resistant variants have a decreased affinity for
Sia2-6Gal-containing sialylglycoconjugates.
Unlike horse serum,
pig serum did not contain substantial amounts of NA-resistant
inhibitors of the LA/87 virus; the inhibitory activity of the serum
decreased 30-fold after NA treatment (Fig. 1). We found that four pig
serum-resistant variants (LA/87 P, LA/87 H-P, LA/87 HP, and LA/87 HP-R)
clearly differ from the parent virus and from other non-pig
serum-adapted variants (LA/87 H and LA/87 R) in that the former have a
lower affinity for the total inhibitors of pig serum (Fig. 1A and 2,
pig serum). The main neutralizing inhibitor of LA/87 in pig serum is
2-macroglobulin (35). Lectin-binding analysis
has shown that 2-6 linkages are predominant between the sialic acid and
the penultimate sugar residue in the sialyloligosaccharide chains of
2-macroglobulin (35). To determine how
influenza viruses become resistant to this pig serum inhibitor, we
analyzed the receptor-binding specificity of these viruses by using a
panel of receptor analogs (Fig. 2). All of our pig serum-resistant
variants had a lower binding affinity for 6'SLN and 6'SLN-PAA (Fig. 2). This finding is consistent with the data of Gimsa et al.
(12), who showed that pig serum-resistant viruses bind
poorly to Sia2-6Gal-containing erythrocytes, and suggests that
resistant variants escape neutralization by pig serum inhibitor due to
their decreased affinity for Sia2-6Gal sialyloligosaccharide
determinants.
Because all pig serum-resistant variants contain a common amino
acid substitution in their HA, 186SI (Table 1), this mutation must be responsible for the decreased binding of Sia2-6Gal and, therefore, for the resistance. In X31 HA, the amino acid at position 186 does not directly contact the sialic acid moiety (Fig. 3), nor does
it directly interact with the 2-3- and 2-6-linked asialic parts of
sialyloligosaccharides (9, 36). The side chain of this amino
acid does, however, contact amino acid residues at positions 190 and
228, which form hydrogen bonds with the terminal hydroxyl group of the
polyhydroxyl tail of sialic acid (36, 41) (Fig. 3). Homology
modeling suggests that the substitution of Ser by the bulkier Ile would
move the side chains of amino acids 190 and 228 inside the pocket
toward the C9-OH group of Neu5Ac (24a). It is not
clear at present how this change could decrease the
binding of Neu5Ac2-6Gal-terminated receptor analogs without
substantially decreasing the binding of free Neu5Ac and Neu5Ac2-3Gal-terminated receptors (Fig. 2).
The inhibitor in rabbit serum is a mannose-binding lectin.
Neither rabbit serum-resistant variant LA/87 R nor LA/87 HP-R
showed any decrease in its affinity for the total
sialylglycoprotein inhibitors in rabbit serum compared with
its parent virus, LA/87 or LA/87 HP, respectively (Fig. 1A; compare
rabbit serum KMT values for LA/87 and LA/87 HP
to those for LA/87 R and LA/87 HP-R). In addition, the affinity of
LA/87 R and LA/87 HP-R for NA-treated rabbit serum was unchanged (Fig.
1B; compare rabbit serum KNA values for LA/87
and LA/87 HP to those for LA/87 R and LA/87 HP-R). Because rabbit
serum-resistant variants showed no change in affinity for the and
inhibitors, it seems likely that these inhibitors were not
responsible for the selection of these variants. We therefore hypothesized that rabbit serum contains a thermostable inhibitor, which was not completely inactivated during the standard heat treatment
procedure (30 min, 56°C) that we used (34). Two lines of
evidence indirectly supported this suggestion. First, both rabbit
serum-resistant strains lost a potential glycosylation site at
Asn246 (Table 1; Fig. 3). This is a typical feature of resistant mutants selected in the presence of inhibitors (17, 18). Second, one of the known serum inhibitors, bovine
conglutinin, is relatively thermostable (17). To examine the
presence of heat-stable -inhibitory activity, the sera were
incubated at 56°C for 30 min and then treated with sodium periodate
to destroy sialic acid-containing inhibitors (2, 21). The
hemagglutination inhibition (HAI) titers of horse, pig, and rabbit
sera treated in this way were 16, 16, and 256. Therefore, a
substantial nonsialoside-mediated inhibitory activity was present
in the heat-inactivated rabbit serum.
Studies by Anders and colleagues (2, 3, 17, 18) identified
inhibitors from bovine, guinea pig, and mouse sera as
calcium-dependent mannose-binding lectins. We therefore examined whether the rabbit serum inhibitor might also be a mannose-binding lectin. As shown in Fig. 4A, the
inhibitory activity of the rabbit preparation was abrogated in the
presence of EDTA and restored by the addition of Ca2+ but
not Mg2+ ions. The HAI activity of the rabbit serum inhibitor was blocked in the presence of the monosaccharides
D-mannose, L-fucose, and N-acetyl-D-glucosamine but
unaffected by D-galactose and
N-acetyl-D-galactosamine (Fig. 4B).
These features indicate that the residual -inhibitory activity of
heat-treated rabbit serum was mediated by a mannose-binding calcium-dependent lectin. Finally, we examined the activity of the
rabbit serum inhibitor against viruses in the HAI test. The
variants LA/87 R and LA/87 HP-R selected in the presence of this
serum were substantially less sensitive to the rabbit serum inhibitor than were LA/87 and the other variants (Fig. 4C). This
finding supports our contention that the inhibitor is responsible for the selection of these serum-resistant variants. Both variants have
lost a glycosylation site at the tip of their HA, which is consistent
with the known mechanism of influenza virus resistance to inhibitors (17, 18).
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FIG. 4.
Properties of the inhibitor present in rabbit serum
after heat inactivation for 30 min at 56°C and periodate treatment.
(A) Effect of bivalent ions on the ability of the preparation to
inhibit the binding of the fetuin (fet)-HRP conjugate to LA/87 virus
(see Materials and Methods for assay details). Serum was diluted in
0.05 M TBS (pH 7.3) (closed circles), TBS supplemented with 5 mM EDTA
(TBS-E) (open circles), or TBS-E containing either 25 mM
CaCl2 (triangles) or 25 mM MgCl2 (solid
squares). (B) Sensitivity of the HAI activity of the inhibitor to
competitive blocking by monosaccharides. The ordinates show the minimal
concentrations of the sugars D-mannose (Man),
L-fucose (Fuc),
N-acetyl-D-glucosamine (GlcNAc),
D-glucose (Glc), D-galactose (Gal), and
N-acetyl-D-galactosamine (GalNAc) that are
required to completely inhibit the HAI activity of 4 HAI units of the
inhibitor. The assay was performed in microplates on 4 HAI units of
LA/87 virus and 0.5% chicken erythrocytes as described previously
(17). No inhibition was observed at the highest
concentration (200 mM) of Gal or GalNAc used. (C) HAI activity of the
inhibitor against LA/87 and its variants. Four HAI units of the
viruses and 0.5% chicken erythrocytes were used for the assay.
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Virus-binding affinity to CAM cells.
The data presented above
indicate that all serum-resistant variants differ from the parent virus
by their decreased affinity for serum inhibitors (either [pig
serum], [horse serum], or [rabbit serum]. Although this
effect alone could account for the resistance, we wanted to know
whether an increased affinity of the mutants for target cells might
additionally help the virus to escape serum inhibitors. To this end,
the relative affinity of the viruses for CAM cells was estimated by the
method of Yewdell et al. (42). Plasma membranes prepared
from CAM cells were adsorbed to wells of microtiter plates and treated
with increasing concentrations of V. cholerae NA to
gradually decrease the density of sialyloligosaccharide receptors.
Virus binding to these cell membranes was then assayed. This assay
relies on the facts that the higher the NA concentration used, the
lower the receptor density left on the cell surface, and that the
viruses that bind to the membranes with lower receptor density have a
higher affinity for the receptor. We found that all of the variants
demonstrated similar or higher affinities for CAM cell membranes than
did the parent virus (Fig. 5); LA/87 R,
LA/87 HP-R, and LA/87 H-P variants had the highest affinities. The
enhancement of virus affinity for cells is thought to promote virus
escape from neutralization by antibodies (8, 22, 39, 42).
Therefore, the higher affinity of LA/87 H-P, LA/87 R, and LA/87 HP-R
for CAM cells may contribute to their serum-resistant phenotypes in
addition to reduced virus binding to the serum inhibitors. However, the
possibility remains that the enhanced binding of these serum-resistant
variants to CAM cells is merely a consequence of the selection for
reduced virus binding to serum inhibitors.
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FIG. 5.
Effect of V. cholerae NA treatment of plasma
membranes of chicken embryo CAM cells on the binding of LA/87 and its
variants.
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We recently showed that CAM cells possess predominantly
Sia2-3Gal-terminated oligosaccharides on their surface (20).
What is the molecular basis for the significantly enhanced affinity these three virus variants have for those receptors? Variant LA/87 H-P
displays increased affinity for free Neu5Ac and 3'SL (Fig. 2). It may
be concluded, therefore, that the higher affinity of this variant for
CAM cells is provided by enhanced interactions with the
sialyloligosaccharide part of the receptors. This enhancement must
be due to mutations 137YD and 186SI, given that these are the
only amino acid differences between LA/87 H and LA/87 H-P (Table 1).
Two rabbit serum-resistant variants, LA/87 R and LA/87 HP-R, differ
from the other viruses in that they lack a carbohydrate chain at
position 246 of the HA close to the receptor-binding site of the
adjacent HA monomer (Fig. 3). Although neither variant exhibited any
substantial change in affinity for sialic acid or monovalent
sialyloligosaccharides compared to the parent virus, both variants
demonstrated dramatic increase in their affinity for polymeric 3'SL
(Fig. 2, 3'SL-PAA). A similar binding pattern has been reported
for the egg-adapted variants of influenza B virus (11),
where the loss of a glycosylation site at Asn187, which is
associated with egg adaptation of the virus, increases the virus's
affinity for Sia2-3Gal-containing receptors without significantly
changing its ability to bind small sialosides. In this case, the
carbohydrate chain at position 187 is thought to sterically interfere
with the macromolecular part of Neu5Ac2-3Gal-terminated macromolecular
receptors (11). Because the carbohydrate moiety at
Asn246 must be relatively close to the carbohydrate moiety at Asn187 of the adjacent HA monomer (Fig. 3), this
mechanism could also operate in the case of LA/87 HA variants.
The receptor-binding phenotype of avian influenza virus changes
when the virus is naturally introduced into horses.
We have shown
that for influenza viruses to grow in eggs in the presence of horse
serum, they must avoid binding to the NA-resistant 4-O-acetylated
sialic acid moieties of EM. To determine whether the same
mechanism operates in nature, we compared the affinities for NA-treated
EM of H3N8 avian influenza viruses, currently circulating H3N8 equine
viruses (so-called equine type 2 viruses), and virus strains
isolated from horses during the 1989 to 1990 influenza outbreak in
China that was caused by the introduction of an influenza virus from
birds (15) (Table 2). The type 2 equine viruses differed
from the avian viruses in that they bound weakly to NA-treated EM in
terms of both absolute binding affinity (KNA)
and relative affinity compared to the mock-treated EM
(KMT/KNA). Of interest, the earliest isolate from the equine influenza outbreak,
A/equine/Jilin/89, exhibited a binding phenotype similar to that of
avian viruses, whereas the phenotype of a strain isolated a year
later (A/equine/Heilonjiang/90) was more like that of type 2 equine
viruses. These findings indicate that there is a pressure in horses to
select viruses that do not bind 4-O-acetylated sialic acid.
Because in the LA/87 virus an amino acid at position 145 contributes to
the recognition of the 4-O-acetyl substituent of sialic acid, we
compared the H3 HA sequences of avian and equine viruses (5). All avian viruses possess 145S, whereas type 2 equine viruses have 145D, which is also found in LA/87 variants adapted to grow in the presence of horse serum. It can be suggested, therefore, that the same molecular mechanism operated during the adaptation of the
avian virus HA in horses. In accord with this notion, the HA of
A/equine/Jilin/89 virus, which displays an avian-like binding phenotype, bears 145S (15). To determine the amino acid
substitutions responsible for the change in receptor-binding phenotype
of A/equine/Heilonjiang/90, we sequenced the HA of this isolate.
Five amino acid substitutions (at positions 48, 91, 190, 216, and 261)
distinguish the HA1 sequence of A/equine/Heilonjiang/90 from that of
A/equine/Jilin/89. Of these substitutions, the mutation 190EK in the
HA of A/equine/Heilonjiang/90 appears to be primarily involved in the
alteration of the receptor-binding properties of the virus. The side
chain of 190E interacts with the glycerol tail of the sialic acid
moiety in the receptor-binding site (36, 41), and 190E
is strictly conserved among all avian influenza viruses
(28). The substitution EK at position 190 probably
moves the sialic acid residue toward the "right" edge of the
receptor-binding site, leading to steric interference between the
4-O-acetyl group and side chain of 145S (Fig. 3).
Because A/equine/Jilin/89 virus, which binds 4-O-acetylated sialic acid
as efficiently as avian viruses, replicated and caused significant
disease in horses, the presence of this sialic acid species does not
appear to pose an impenetrable barrier for transmission of the avian
virus to horses. Rather, this sialic acid species appears to exert a
selective pressure that leads to distinct changes in the
receptor-binding phenotype of the HA of the virus during its further
adaptation in this host. It may be that 2-macroglobin or
some other glycoproteins containing 4-O-acetylated
sialyloligosaccharides present in horse respiratory secretions are
responsible for this selection. It is also possible that 4-O-acetylated
sialic acids are present on susceptible cells in horses and that
selection is due to the inability of viral NA to remove these sialic
acid residues from oligosaccharides and to provide the release of the virus from these cells or to prevent virus self-aggregation. In either
event, selection stems from the presence of NA-resistant 4-O-acetylated
sialylglycoconjugates in horses. Although we do not know if natural
receptor analog inhibitors have played a role in other events of
interspecies transmission of influenza virus, the present study
demonstrates, for the first time, that a species difference in sialic
acid contributes to the evolution of influenza viruses.
We are very grateful to N. V. Bovin and A. B. Tuzikov,
Shemyakin Institute of Bio-Organic Chemistry, Moscow, Russia, and
V. E. Piskarev, Nesmeyanov Institute of Organoelement
Compounds, Moscow, Russia, for providing sialylglycopolymers and 6'SLN,
respectively. We thank R. Bethel of Glaxo Wellcome for providing the NA
inhibitor GG167, Krisna Wells for technical assistance, and Susan
Watson for editing the manuscript.
This work was supported by Public Health Service research grants
AI-33898 from the National Institute of Allergy and Infectious Diseases, by the CAST program from the National Research Council, by
Cancer Center Support (CORE) grant CA-21765, and by the American Lebanese Syrian Associated Charities. M. Matrosovich was
supported by a Karnofsky fellowship from St. Jude Children's Research
Hospital.
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Abstract
Introduction
