﻿JOURNAL OF VIROLOGY, Aug. 2003, p. 8418­8425 Vol. 77, No. 15
0022-538X/03/$08.000 DOI: 10.1128/JVI.77.15.8418­8425.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Overexpression of the -2,6-Sialyltransferase in MDCK Cells Increases
Influenza Virus Sensitivity to Neuraminidase Inhibitors
Mikhail Matrosovich,1,2
* Tatyana Matrosovich,1
Jackie Carr,3
Noel A. Roberts,3
and
Hans-Dieter Klenk1
Institute of Virology, Philipps University, 35037 Marburg, Germany1
; M.P. Chumakov Institute of Poliomyelitis and Viral
Encephalitides, Moscow 142782, Russia2
; and Roche Products Ltd., Welwyn Garden City,
Herts AL7 3AY, United Kingdom3
Received 24 February 2003/Accepted 15 May 2003
No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to
neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration
of sialyl-2,6-galactose (Sia[2,6]Gal)-containing virus receptors in conventional cell lines, replication of
human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of
Sia(2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity
to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells
expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids
than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis
agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased,
whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The
sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was
substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neur-
aminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably
transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.
The neuraminidase (NA) of influenza A and B viruses
cleaves the -glycosidic linkages between sialic acid and the
adjacent sugar and thus destroys virus receptors on the cell
surface, extracellular inhibitors, and viral glycoproteins (re-
viewed in references 2 and 8). The NA activity is believed to be
particularly important at the late stages of infection by pre-
venting hemagglutinin (HA)-mediated self-aggregation and fa-
cilitating release of progeny virions from cells. Interaction of
virions with cell-associated and soluble sialylglycoconjugates of
the host is mediated by HA and NA in an antagonistic manner,
which has to be carefully balanced to allow efficient virus rep-
lication (reviewed in reference 36).
With increasing use of neuraminidase inhibitors (NAI) for
influenza treatment, there is a need for a suitable methodology
to monitor for emergence of virus resistance (32, 34, 38). In
cell culture experiments, resistance to NAI results from muta-
tion of either HA, NA, or both glycoproteins. Mutations in HA
usually precede NA mutations and reduce virus affinity for
sialic acid-containing receptors, thereby decreasing the depen-
dency of the virus on NA function, whereas mutations in NA
decrease the binding affinity of the inhibitor to the catalytic site
(reviewed in references 19, 29, and 30). In a clinical setting,
NA-mediated resistance seems to be the primary mechanism
of resistance to NAI and can be easily and reliably monitored
using an in vitro enzyme inhibition assay (32, 34, 38). Since the
possibility cannot be excluded that the loss of sensitivity to NAI
in humans occurs also as a result of HA mutations (18, 20), it
is necessary to develop techniques to study this type of resis-
tance in low-passage-number clinical isolates.
The method of choice for testing virus sensitivity to NAI
would be a virus neutralization assay in cell culture that ac-
counts for both HA- and NA-mediated resistance. However,
there is no good correlation between virus sensitivity to NAI in
vivo and in laboratory cell cultures. The sensitivity of clinical
virus isolates to NA inhibitors can vary in cell culture assays
dramatically (up to complete insensitivity) despite a uniform
high sensitivity of the enzyme in NA-inhibition tests (1, 3,37).
This problem is likely due to a mismatch between virus recep-
tors in humans and in available cell culture systems. The target
cells for virus replication in human airway epithelium express
high concentrations of Sia(2,6)Gal-containing receptors and
small amounts of Sia(2,3)Gal-containing receptors (below ab-
breviated to 6-linked and 3-linked sialic acid receptors, respec-
tively) (4, 9). Clinical isolates of human influenza viruses bind
strongly to 6-linked sialic acids but do not bind to 3-linked
sialic acids (references 13 and 21 and references therein). It is
therefore believed that in order to adequately assay human
influenza virus sensitivity to NAI, a cell line is required which
supports efficient growth of clinical influenza virus isolates and
expresses large amounts of 6-linked sialic acids and small
amounts of 3-linked sialic acids (38). Unfortunately, the con-
centration of 6-linked sialic acids in continuous cell lines used
for propagation of influenza viruses in the laboratory (such as
MDCK and VERO cells) is relatively low and is comparable to
the concentration of 3-linked sialic acids (16, 21, 33).
In this study, we wished to test whether performance of
standard laboratory cells in the NAI sensitivity assay can be
* Corresponding author. Mailing address: Institute of Virology,
Philipps University, Robert Koch str. 17, 35037 Marburg, Germany.
Phone: 49-(6421)-286-5166. Fax: 49-(6421)-286-8962. E-mail: mikhail
.matrosovich@med.uni-marburg.de.
8418
improved by purposefully changing the concentration of virus
receptors on the cell surface. To this end, we permanently
transfected MDCK cells with the gene of the human CMP-N-
acetylneuraminate beta-galactoside -2,6-sialyltransferase
(SIAT1) [EC 2.4.99.1; other acronyms used in the literature
are ST6Gal I and ST6(N)], an enzyme that catalyzes the -2,6-
sialylation of N-acetyllactosamine moieties of glycoproteins
and glycolipids (see references 11 and 35 for a review). Based
on previous reports on successful overexpression of SIAT1 in
mammalian cells (6, 10, 12, 24, 25), we expected that transfec-
tion with SIAT1 would increase -2,6-sialylation of the cellular
and viral glycoconjugates and, thus, would increase a virus
dependency on a neuraminidase activity and, hence, sensitivity
to NA inhibitors. Furthermore, we hoped that transfection
would decrease -2,3-sialylation in the cells due to a competi-
tion between SIAT1 and 2,3-specific sialyltransferases for the
same precursor substrate [Gal(1,4)GlcNAc-R] (6, 24).
MATERIALS AND METHODS
Viruses. Clinical isolates in MDCK cells of A/Memphis/14/96 (H1N1) and
B/Memphis/25/99 at passage level 2 were kindly provided by Robert Webster (St.
Jude Children's Hospital). The Memphis/14/96 virus was plaqued in MDCK
cells; two plaques of different size were picked, amplified in MDCK cells, and
designated variants M and M1. In parallel, the original passage 2 virus was
passaged twice in embryonated chicken eggs. The virus progeny from two dif-
ferent eggs were plaque purified in MDCK cells, amplified in MDCK cells, and
designated variants E and E1. The complete HA genes of the variants were
sequenced by the dideoxy method, using an automatic sequencer (Perkin-
Elmer). The GenBank accession numbers for the sequences are from AY282756
to AY282759.
A clinical isolate of A/Sydney/5/97-like virus (H3N2) isolated and propagated
in MDCK cells and its oseltamivir-resistant variant with an R292K mutation in
NA obtained from a patient on the treatment arm of a phase III efficacy study for
oseltamivir phosphate were described previously (7). A clinical isolate of A/Wu-
han/359/95-like virus (H3N2) and its oseltamivir-resistant variant with mutation
E119V in NA were described previously (22).
MDCK cells. The MDCK cells used were originally obtained from Alan Hay
at the National Institute of Medical Research, London, United Kingdom, and
passaged in Frederick Hayden's Laboratory at the University of Virginia Health
Sciences Center, using standard laboratory procedures prior to distribution to
Roche Discovery Welwyn. In this study, the original and SIAT1-transfected cells
were passaged in Dulbecco's modified Eagle medium (Gibco BRL) supple-
mented with 10% fetal calf serum.
Expression vector pIRESneo-SIAT1 and stable transfection of MDCK cells.
The cDNA clone of human SIAT1 in the pcDNA3.1GS expression vector was
obtained from the GeneStorm human clone collection (GeneStorm clone H-
X17247 M; Invitrogen). The full-length open reading frame of SIAT1 was PCR
amplified with primers 5GGCGCAATAGCGGCCGCGCCACCATGATTCA
CACCAACCTGAAG-3 and 5GGCCGATATGGATCCTTAGCAGTGAAT
GGTCCGGAAGCCAG-3, containing restriction sites for NotI and BamHI,
respectively. The PCR product was cloned into the pIRESneo bicistronic expres-
sion vector (Clontech) at 5 NotI and 3 BamHI. Sequencing of the initially
prepared construct revealed a single-nucleotide deletion in the coding region of
SIAT1, leading to a frame shift (plasmid pIRESneo-SIAT1-del). The deletion
was located in the region complementary to the 5-end PCR primer used and was
most likely caused by an error during primer synthesis. We corrected the deletion
by using a QuikChange site-directed mutagenesis kit (Stratagene).
MDCK cells were transfected with pIRESneo-SIAT1 and pIRESneo-SIAT1-
del plasmids using Lipofectamine 2000 (Life Technologies) according to the
manufacturer's instructions. Transfected cultures were cultivated in the presence
of 1.5 mg of antibiotic G418 sulfate (Promega)/ml. After 2 weeks, cells surviving
the selection were pooled, passaged three times in 1.5 mg of G418/ml, and frozen
in aliquots. Serial passages of stably transfected cells were done in the presence
of 1 mg of G418/ml. Terminal plating of the cells for the infection experiments,
isolation of plasma membranes, and lectin staining were performed without
G418. The cells in these experiments were used at passage levels from 5 to 30
starting from the frozen cell stock.
Detection of 6-linked and 3-linked sialic acids. Cell surface expression of sialic
acids in two different types of linkages was quantified by using digoxigenin
(DIG)-labeled lectins Sambucus nigra agglutinin (SNA) specific for 6-linked
sialic acids, Maackia amurensis agglutinin (MAA) specific for 3-linked sialic
acids, and either fluorescein isothiocyanate-labeled or peroxidase-labeled anti-
DIG antibodies from the DIG-glycan differentiation kit (Boehringer Mannheim,
Mannheim, Germany). Fluorescence-activated cell sorter (FACS) analysis of the
cells stained with lectins was performed as described previously (17) using a
FACScan fluorospectrometer (Becton Dickinson). For the solid-phase assay of
lectin binding, plasma membranes were isolated from MDCK and MDCK-
SIAT1 cells as described previously (14). Membrane preparations were sus-
pended in phosphate-buffered saline (PBS) to a final protein concentration of 2
g/ml, and 0.05-ml aliquots were incubated in the wells of a polystyrene 96-well
microplate overnight at 4°C. Wells incubated with PBS served as a control of the
background binding. The plate was washed with PBS and blocked with 0.1 ml of
a 0.04% solution of bovine serum albumin (BSA) in PBS for 1 h at 37°C.
Solutions (0.05 ml each) of DIG-labeled lectins (either SNA [2 g/ml] or MAA
[5 g/ml]) in lectin-binding buffer (LBB) (0.2% BSA, 1 mM Mg2
, Ca2
, and
Mn2
in Tris-buffered saline [pH 7.2]) were incubated in the wells for 2 h at 4°C.
After washing with PBS, the plate was incubated with 0.05 ml of peroxidase-
labeled anti-DIG antibodies/well in LBB for 1 h at 4°C. O-phenylenediamine was
used as a chromogenic substrate.
Binding of virus to cell membranes. Virus attachment to cell membranes was
assayed by using the microplate adsorption method (14). In brief, viruses were
clarified by low-speed centrifugation, pelleted by high-speed centrifugation
through a 25% sucrose cushion, and resuspended in 0.1 M Tris-HCl buffer (pH
7.3). Ninety-six-well plates coated with the cell membranes and blocked as
described above were incubated with 0.05 ml of serially diluted viruses in 0.5%
BSA-PBS for 30 min at 37°C. The plates were washed with ice-cold washing
solution (PBS-T) (0.02% Tween 80 in PBS) and incubated with sheep antiserum
against influenza virus A/Taiwan/1/86 (H1N1) (Centers for Disease Control and
Prevention, Atlanta, Ga.) for 1 h at 4°C, followed by washing (PBS-T) and
incubation with peroxidase-labeled anti-sheep antibodies (DAKO) for an addi-
tional hour at 4°C. PBS containing 1% BSA and 0.05% Tween 80 was used for
the preparation of working dilutions of immunoreagents. The amount of bound
conjugate, which reflected the amount of the virus present in the wells, was
quantified using o-phenylenediamine as a substrate. The absorbency at 490 nm
was measured, and the data were converted to Scatchard plots (A490 versus
A490/C), where the concentration of the viruses (C) was expressed in hemagglu-
tination units. The background virus binding to the noncoated blocked wells did
not exceed 0.02 optical units and was disregarded.
Peroxidase-labeled resialylated fetuin. To prepare monospecific fetuin-horse-
radish peroxidase (HRP) conjugates containing either 6-linked- or 3-linked sialic
acid moieties, bovine asialofetuin (Sigma, St. Louis, Mo.) was first conjugated
with HRP by using the periodate method (5). Solutions in phosphate buffer (0.1
M, pH 7.0) containing 8 mg of asialofetuin-HRP conjugate/ml, 1.5 mM CMP-
Neu5Ac (Boehringer), 2 mM MgCl2, and either 80 mU of -2,6-sialyltransferase
from rat liver (WAKO Chemicals)/ml or 8.5 mU of rat recombinant -2,3-(N)-
sialyltransferase (Calbiochem)/ml were incubated at 37°C for 20 h. Resialylated
conjugates were dialyzed against 0.1 M Tris-HCl buffer (pH 7.3), diluted with an
equal volume of glycerol, and stored at 20°C.
Binding affinity assay. The binding of the HRP-labeled fetuins to the viruses
was studied using a solid-phase assay, as described previously (27). In brief,
viruses adsorbed in the wells of 96-well EIA microplates (Greiner) were incu-
bated with serial dilutions of HRP-labeled resialylated fetuins in PBS supple-
mented with 0.02% BSA, 0.02% Tween 80, and a 2 M concentration of the
neuraminidase inhibitor oseltamivir carboxylate. After incubation for 1 h at 4°C,
the plates were washed with ice-cold PBS-T, and the amount of bound labeled
fetuin-HRP was quantified by the peroxidase activity present in the wells. The
binding data were converted to A490/C versus A490 Scatchard plots, arbitrarily
taking the concentration of fetuin-HRP stocks for 1,000 U/ml. The association
constants of the virus-fetuin complexes were determined from the slopes of these
plots.
Focus reduction assay. Cell cultures in 96-well plates (Greiner) were inocu-
lated with 0.05 ml of either infection medium (IM) (0.1% BSA in Dulbecco's
modified Eagle medium) or serial 10-fold dilutions of oseltamivir carboxylate in
IM (concentration range from 2 nM to 200 M)/well. After that, 50 l/well of
serial twofold virus dilutions in IM were added to cover a range of multiplicities
of infection (MOIs) from about 5 to 100 focus-forming units per well. TPCK-
trypsin (final concentration, 1 g/ml) was added simultaneously with the virus to
provide for multicycle replication. After 24 to 48 h of incubation at 35°C, the cells
were fixed with 4% paraformaldehyde for 1 h and permeabilized with 0.2%
solution of Triton X-100 in PBS for 20 min. These and all subsequent treatments
VOL. 77, 2003 ALTERATION OF INFLUENZA VIRUS RECEPTORS IN MDCK CELLS 8419
of the cells were performed at room temperature. Fixed cultures were immuno-
stained for the expression of viral nucleoprotein by incubating for 1 h with
monoclonal antibodies specific for the nucleoprotein of either influenza A virus
(kindly provided by Alexander Klimov at Centers for Disease Control) or influ-
enza B virus (Serotec) followed by 1 h of incubation with peroxidase-labeled
anti-mouse antibodies (Sigma) and 30 min of incubation with precipitate-form-
ing peroxidase substrate (True Blue; KPL). Ten-percent horse serum plus 0.05%
Tween 80 in PBS was used for the preparation of working dilutions of immu-
noreagents. Stained plates were scanned on a flatbed scanner with the data
acquired by Adobe Photoshop 7.0 software. To compensate for some occasional
variation in the size and number of foci in individual wells, we used two to four
replicate wells per experimental point. Ninety percent inhibitory concentrations
presented in Table 1 were estimated by visual observation of cultures infected at
a MOI of 20 to 40 focus-forming units per well. These values correspond to the
lowest of the drug concentrations used, which reduced the stained area in the
wells at least 10-fold.
RESULTS
Transfection of MDCK cells with SIAT1 alters surface ex-
pression of 6-linked and 3-linked sialic acids and enhances
binding of a human influenza virus. For the permanent ex-
pression of 2,6-sialyltransferase, we cloned human SIAT1
cDNA into the bicistronic mammalian expression vector
pIRESneo. Because this vector expresses the gene of interest
and an antibiotic resistance marker from the same mRNA,
nearly all colonies surviving the selective pressure must stably
express the gene of interest (31). Therefore, after transfection
of MDCK cells with the pIRESneo-SIAT1 plasmid, we did not
isolate individual resistant clones but pooled several clones
which survived the selection with G418 sulfate.
To compare the expression of 3-linked and 6-linked sialic
acids on the surface of transfected and parent cells, we used
the linkage-specific lectins SNA and MAA. FACS analysis
indicated that transfection with the pIRESneo-SIAT1 plasmid
enhanced expression of 6-linked sialic acids and simulta-
neously lowered expression of 3-linked sialic acids (Fig. 1, top
panel). In a solid-phase lectin binding assay, the binding of
SNA to the plasma membrane of transfected cells increased
about twofold, and the binding of MAA decreased by about
50% (Fig. 1, bottom panel). This pattern of reactivity with
lectins remained unchanged after 25 passages of MDCK-
SIAT1 cells in the presence of G418 (data not shown), indi-
cating that the sialylation phenotype of the cells was stable. As
a control, we used MDCK cells permanently transfected with a
pIRESneo-SIAT1-del plasmid which contained a frame shift
deletion in the SIAT1 gene (see Materials and Methods). In
this case, as expected, the lectin binding assay revealed no
differences between parent and transfected cells (data not
shown).
To test whether changes in sialylation of SIAT1-transfected
cells affected the strength of virus binding to these cells, we
used two influenza viruses with distinct receptor-binding phe-
FIG. 1. Binding of the linkage-specific lectins SNA and MAA to
cell surface receptors of SIAT1-transfected and parent MDCK cells.
Top panel, MDCK-SIAT1 cells (shaded profiles) and MDCK cells
(open profiles) were incubated with DIG-labeled lectins followed by
incubation with fluorescein isothiocyanate-labeled anti-DIG antibod-
ies and subjected to FACS analysis. Bottom panel, preparations of cell
plasma membranes adsorbed in the wells of a 96-well plate were
incubated with DIG-labeled lectins followed by incubation with per-
oxidase-labeled anti-DIG-antibodies. The data represent ratios of ab-
sorbencies in the wells coated with membranes of MDCK-SIAT1 cells
to those in wells coated with membranes of MDCK cells.
TABLE 1. Receptor-binding characteristics and sensitivities to oseltamivir carboxylate of HA variants of A/Memphis/14/96 (H1N1)
Variant
Amino acid
substitution
in the HAa
Binding affinityc
, Kass (ml/U)
Sensitivity to oseltamivir
carboxylate, IC90 (M)d
2,6-Fetuin-HRP 2,3-Fetuin-HRP 24 h 48 h
M 3.3  0.10 0.046  0.021 0.1 1.0
M1 E156K 4.9  0.58 0.70  0.30 NDe
0.01
E N129Kb
4.8  0.60 0.48  0.20 0.01 NTf
E1 Q226R 0.09  0.02 0.85  0.31 100 100
a
With respect to HA sequence of variant M. H3 numbering system.
b
The mutation destroys a glycosylation sequon in the HA.
c
Affinity of virus binding to HRP-labeled resialylated fetuins containing either 6-linked or 3-linked sialic acid moieties. The higher values of association constants
(Kass) reflect higher affinities.
d
Focus reduction assay in MDCK-SIATI cells was performed on two different occasions, with infection times of 24 and 48 h, respectively (see the text). IC90, 90%
inhibitory concentration.
e
ND, not determined. IC90 of MI variant could not be determined due to a poor virus spread 24 h postinfection.
f
NT, not tested.
8420 MATROSOVICH ET AL. J. VIROL.
notypes. The clinical A/Memphis/14/96-M (H1N1) isolate dis-
played a preference for 6-linked sialic acids and did not bind to
3-linked sialic acids as is typical for human viruses. The egg-
adapted variant E1 of this virus had the opposite receptor
specificity due to a point mutation Q226R in the HA (see
Table 1). The M virus bound to membranes isolated from
MDCK-SIAT1 cells significantly better than it bound to mem-
branes of parent MDCK cells (Fig. 2, top panel). On the
contrary, the egg-adapted mutant bound to transfected cells
more weakly than to MDCK cells (Fig. 2, bottom panel), al-
though the difference in binding was less pronounced than in
the case of M virus.
Transfection of MDCK cells with SIAT1 increases sensitiv-
ity of influenza viruses to oseltamivir carboxylate in a cell
culture neutralization assay. Because influenza viruses did not
produce clearly visible plaques in MDCK-SIAT1 cells, we used
a focus reduction assay to compare virus sensitivity to NA
inhibitors in the parent MDCK cells and in the SIAT1-trans-
fected cells. Parallel cultures of two cell lines in 96-well plates
were infected with serial virus dilutions in the presence or
absence of oseltamivir carboxylate and immunostained for the
expression of virus nucleoprotein. The effect of the drug on
virus spread in parent and transfected cells was compared at
MOIs which resulted in growth of about 15 to 50 foci of
infected cells per well. In the first series of experiments, three
clinical virus isolates, A/Sydney/5/97 (H3N2), A/Memphis/14/
96-M (H1N1), and B/Memphis/25/99, were tested. All three
viruses displayed a higher sensitivity to oseltamivir carboxylate
in MDCK-SIAT1 cells than in nontransfected cells. This pat-
tern of sensitivity was reproducible in replicate assays per-
formed on different days. Results of one representative exper-
iment are shown in Fig. 3. In MDCK-SIAT1 cells, 0.001, 0.01,
and 0.1 M concentrations of the drug markedly decreased the
size of infectious foci formed by H3N2, H1N1, and type B
viruses, respectively. A further 10-fold increase in the respec-
tive drug concentrations completely abolished virus spread in
transfected cells, as judged by the disappearance of the comet-
like foci of infection. By contrast, even the highest drug con-
centrations did not completely inhibit formation of foci in
MDCK cells. Thus, MDCK-SIAT1 cells provide a more sensi-
tive system than MDCK cells for testing the susceptibility of
human influenza A and B viruses to NAI.
To check whether a focus reduction assay with MDCK-
SIAT1 cells could clearly identify viruses with drug-resistant
mutations in NA, we compared two drug-sensitive clinical virus
isolates with their oseltamivir treatment-selected drug-resis-
tant mutants (Fig. 4). A 0.1 M concentration of oseltamivir
carboxylate substantially inhibited spread of a wild-type A/Syd-
FIG. 2. Scatchard plots for the binding of A/Memphis/14/96-M
(H1N1) (top panel) and its egg-adapted mutant E1 (bottom panel) to
plasma membranes from MDCK cells (closed boxes, solid line) and
from MDCK-SIAT1 cells (open circles, dotted line). Data points rep-
resent results of two replicate experiments performed on the same
plate as described in Materials and Methods.
FIG. 3. Effect of oseltamivir carboxylate on the formation of virus
foci in parallel cultures of MDCK (M) and MDCK-SIAT1 cells (ST)
infected with the same virus multiplicity. Experiments were performed
in 96-well plates as described in Materials and Methods using clinical
virus isolates A/Sydney/5/97 (H3N2) (top panel), A/Memphis/14/96-M
(H1N1) (middle panel), and B/Memphis/25/99 (bottom panel). The
infection time was 24 h for the type A viruses and 48 h for the type B
virus.
VOL. 77, 2003 ALTERATION OF INFLUENZA VIRUS RECEPTORS IN MDCK CELLS 8421
ney/97-like virus (Fig. 4a). By contrast, a 100-fold-higher con-
centration of the drug (10 M) was required for a detectable
inhibition of the drug-resistant variant with an R292K muta-
tion in NA. Interestingly, the infectious foci of the mutant in
the absence of the drug were smaller that those of the wild-type
virus, suggesting that the NA mutation impaired spread of the
virus in MDCK-SIAT1 cells. This effect is consistent with the
reported compromised infectivity of the R292K NA mutant in
vivo (7). Because of a slower spread of the NA mutant, this
virus produced very small foci 24 h postinfection, making it
impossible to assess a reduction in focus size in the presence of
NA inhibitor (data not shown). To solve this problem, we used
longer infection times for comparison of wild-type viruses and
NA mutants. It has to be mentioned that an increase in the
incubation time resulted in an apparent decrease in the virus
sensitivity to the inhibitor. For example, 0.001 and 0.1 M
concentrations of the drug were required, respectively, for a
substantial inhibition of wild-type virus spread during 24 h
(Fig. 3) and 40 h of infection (Fig. 4a). Thus, standardization of
the assay time will be important for routine drug sensitivity
monitoring using a focus reduction assay.
Figure 4b shows the results obtained with another pair of
pre- and posttreatment H3N2 viruses which were tested in
parallel in two cell lines. In MDCK cells, neither virus is drug
sensitive, making it impossible to discriminate between resis-
tant mutant and the original virus. However, in MDCK-SIAT1
cells, the wild-type virus can be clearly differentiated from its
drug-resistant mutant with an E119V mutation in the NA.
Thus, experiments with two pre- and posttreatment virus
pairs tested suggested that NA-mediated drug resistance can
be reliably detected in SIAT1-transfected cells.
Sensitivity to NA inhibitor in MDCK-SIAT1 cells correlates
with virus affinity for 6-linked sialic acids. To address the
question of whether HA-mediated drug resistance could be
adequately assayed in MDCK-SIAT1 cells, we needed a panel
of virus variants which differ by the receptor-binding charac-
teristics of their HAs. To generate such variants, we selected
two receptor-binding mutants (E and E1) of the clinical H1N1
virus isolate A/Memphis/14/96 by adaptation of the virus to
embryonated hen's eggs. Furthermore, during the plaque pu-
rification of the original human virus in MDCK cells, two
MDCK-derived variants were obtained, M and M1. The com-
plete HA genes of four virus variants were sequenced, and
their receptor-binding characteristics were determined using
resialylated fetuin, which carried either 3- or 6-linked sialic
acids (Table 1).
Among both MDCK-derived viruses, only variant M dis-
played a typical receptor-binding phenotype of clinical human
isolates, namely, a high affinity for 6-linked sialic acids and a
lack of binding to 3-linked sialic acids (13, 15). Variant M1
differed from variant M by the amino acid substitution E156K,
which increased a positive charge of the globular head of HA
by two units of charge and enhanced the virus binding affinity
for both 3-linked and 6-linked sialic acids. Analysis of the HA
sequences in the Influenza Sequence Database (26) indicated
that the A/Memphis/14/96 strain used in this study was phylo-
genetically closely related to H1N1 viruses isolated in 1995 to
1996. Among about 30 H1 HA sequences from this time pe-
riod, three sequences carry 156G and the rest carry 156E.
Furthermore, none of more than a hundred human H1N1
viruses isolated between 1995 and 2001 contains 156K. Based
on the results of sequence comparison and on receptor-binding
characteristics, we conclude that variant M represents the orig-
inal clinical human virus, whereas M1 is a receptor-binding
mutant selected in MDCK cells.
Egg adaptation of human viruses is based on selection of HA
mutants with enhanced affinity for 3-linked sialic acids and, as
a rule, leads to accompanying changes in the affinity for
6-linked sialic acids (13, 15, 21). Accordingly, the egg-adapted
variants E and E1 demonstrated significantly enhanced binding
to fetuin carrying 3-linked sialic acids compared to the binding
of the original M variant (Table 1). In the case of variant E, the
HA mutation N129K, responsible for the adaptation, increased
the positive charge of HA, destroyed a glycosylation sequon
near the receptor binding site, and enhanced the virus affinity
for both 3-linked and 6-linked sialic acids. By contrast, substi-
tution Q226R in E1 almost completely destroyed the ability of
the virus to bind to 6-linked receptors. Interestingly, the amino
acid in position 226 is crucial for the recognition of the Sia-Gal
linkage by H2 and H3 subtype HAs (reference 28 and refer-
ences therein). The E1 variant described here provides the first
example of a similar role of the 226 position in an H1N1
human virus.
We next compared four receptor-binding variants for their
sensitivity to oseltamivir carboxylate in MDCK-SIAT1 cells
(Table 1). In the first experiment, the cells were fixed 24 h after
FIG. 4. Comparison of clinical isolates (wt) and their drug-resistant
variants with NA mutations (mut) by focus reduction assay. (a) A/Syd-
ney/5/97-like virus (H3N2) and its variant with NA-substitution R292K
assayed in MDCK-SIAT1 cells. Infection time was 40 h. (b) A/Wuhan/
359/95-like virus (H3N2) and its variant with NA mutation E119V
assayed in parallel in MDCK cells (two upper rows) and in MDCK-
SIAT1 cells (two lower rows). Infection time was 48 h.
8422 MATROSOVICH ET AL. J. VIROL.
infection. Under these conditions, 0.1 and 0.01 M concentra-
tions of the drug were required to inhibit the spread of M and
E variants by 90%, respectively, whereas E1 virus was not
inhibited to any significant extent at the highest concentration
of oseltamivir carboxylate tested (100 M). Because the M1
variant produced very tiny foci 24 h postinfection, precluding
unambiguous detection of inhibition, the assay was repeated
for M, M1, and E1 viruses using a 48-h incubation time. In this
case, the M1 variant was 100-fold more sensitive to the NA
inhibitor than the original M virus. Together these results
indicated that the sensitivity of the viruses to the drug in
MDCK-SIAT1 cells was mainly dependent on virus affinity for
6-linked rather than for 3-linked sialic acid receptors. Namely,
E and M1 mutants with enhanced binding affinity for 6-linked
receptors were more sensitive to oseltamivir carboxylate than
the wild-type virus, whereas the E1 variant was completely
resistant, in correlation with its inability to bind to 6-linked
sialic acids and despite its highest affinity for 3-linked sialic
acids. It seems, however, that the virus affinity for 3-linked
receptors could somewhat modify its susceptibility to the drug.
Thus, a high sensitivity to oseltamivir of the M1 variant com-
pared to that of the E variant correlated with a higher affinity
of the former virus for 2,3-fetuin.
DISCUSSION
Low-passage-number clinical influenza virus isolates often
appear insensitive to NAI when tested in MDCK or other
laboratory cell lines. This precludes the detection of changes in
viral drug sensitivity (resistance) between pre- and posttreat-
ment isolates and can give rise to false-positive identification of
resistance in population screening. This study was undertaken
to test whether a genetically engineered increase in the density
of human influenza virus receptors on the surface of MDCK
cells can make the virus more sensitive to NAI in these cells.
To address this question, we permanently transfected MDCK
cells with the gene of human 2,6-sialyltransferase.
Stable transfection with the SIAT1 gene increased the con-
centration of 6-linked sialic acids on the surface of MDCK cells
about twofold. This effect is consistent with enhanced -2,6-
sialylation in several other mammalian cell lines after their
transfection with SIAT1 (6, 10, 12, 24, 25). We found that a
typical non-egg-adapted human H1N1 influenza virus bound
more strongly to transfected cells than to parent MDCK cells.
This result confirmed that expression of SIAT1 enhanced the
concentration of 6-linked sialic acid receptors accessible to the
virus on a cell surface. SIAT1 catalyses synthesis of 6-sialyl
(N-acetyllactosamine) [Neu5Ac(2,6)Gal(1,4)GlcNAc], the
common high-affinity receptor determinant of human influ-
enza A and B viruses (13). We speculate, therefore, that all
human influenza viruses irrespective of their type and subtype
will likely display enhanced binding to MDCK-SIAT1.
Representative clinical isolates of H1N1 and H3N2 influ-
enza A viruses and type B viruses were substantially more
sensitive to the NA inhibitor oseltamivir carboxylate in SIAT1-
transfected cells than in parent cells. These data support the
hypothesis that the well-known low sensitivity of clinical iso-
lates of human viruses to NAI in MDCK cells is due to a low
expression of 6-linked virus receptors in these cells (19, 30, 38).
There are at least two possible mechanisms by which increased
-2,6-sialylation in the cells enhances virus sensitivity to NAI.
First, due to an increased density of 6-linked cell-surface sialic
acids, progeny virions attach more strongly to the surface of
MDCK-SIAT1 cells and hence require a higher neuraminidase
activity for receptor cleavage and virus release. Second, en-
hanced activity of SIAT1 in the cells can lead to an increased
-2,6-sialylation of the viral glycoproteins. As a consequence, a
higher level of NA activity would be needed to desialylate viral
HA and NA to a degree that prevents self-aggregation of the
progeny virions. Thus, our results suggest that transfection
with SIAT1 may solve or at least reduce the major problem
which hampers utilization of MDCK cells for a neutralization
assay of virus sensitivity to NAI (38).
In addition to the low sensitivity of clinical human viruses to
NAI in standard laboratory cells potentially giving false-posi-
tive resistance results (3, 37), false-negative resistance results
in MDCK cells were also reported (18, 20). For example, an
influenza B virus isolate recovered from an immunocompro-
mised child after 12 days of treatment with zanamivir was
highly sensitive to the drug in MDCK cells despite a drug-
resistant phenotype in the NA enzyme inhibition assay (18).
Compared to the pretreatment isolate, this virus had a drug-
resistant mutation in the NA catalytic site (R152K) and a
mutation in the region of the HA receptor-binding site
(T198I). The HA mutation increased the virus binding to
3-linked receptors and, as a result, to MDCK cells which ex-
press high quantities of 3-linked sialic acids (21, 33). Enhanced
binding to cells increased the virus dependence on the NA
activity, thereby masking the drug-resistant effect of the NA
mutation. These data suggested that laboratory cells do not
adequately mimic virus infection of airway epithelium in vivo,
not only because of a lower level of expression of 6-linked sialic
acids but also because of a higher concentration of 3-linked
receptors. If this hypothesis is correct, a reduced expression of
3-linked sialic acids in laboratory cells would be desirable for
adequate testing of virus sensitivity to NAI. In view of this
hypothesis, we hoped that permanent expression of the SIAT1
gene in MDCK cells could decrease -2,3-sialylation in these
cells due to competition between SIAT1 and -2,3-sialyltrans-
ferases which utilize the same substrate [Gal(1,4)GlcNAc]
(for example, ST3Gal III and ST3Gal IV [35]). The MAA
which we used to determine changes in expression of 3-linked
sialic acids selectively binds to Neu5Ac/Gc(2,3)Gal(1,4)Gl-
cNAc moieties synthesized by ST3Gal III and ST3Gal IV (23).
As we expected, the concentration of these moieties in the
SIAT1-transfected cells decreased by about 50%. However,
because one cannot expect a competition between SIAT1 and
-2,3-sialyltransferases with different substrate specificities
[such as ST3Gal I and ST3Gal II, which sialylate Gal(1,3)
GalNAc], the total decrease in expression of 3-linked sialic
acids in all possible contexts was certainly less than that deter-
mined with MAA. This notion may explain why we observed a
relatively marginal decrease in binding of an egg-adapted hu-
man virus to MDCK-SIAT1 cells.
Although NA mutations appear to be the major mechanism
of influenza virus resistance to NAI in a clinical setting (32, 34,
38), the possibility that resistance emerges due to mutations in
HA cannot be completely excluded. Thus, in a study cited
above, the HA mutation T198I in influenza B virus was first
observed after 8 days of treatment with zanamivir, at least 4
VOL. 77, 2003 ALTERATION OF INFLUENZA VIRUS RECEPTORS IN MDCK CELLS 8423
days before the emergence of a drug resistance mutation in NA
(18). The substitution T198I was found to lower substantially
the HA affinity for 6-linked sialic acid receptors. The authors
suggested that this mutation resulted in a decreased virus bind-
ing to human airway epithelial cells (where 6-linked receptors
predominate) and thus reduced dependence of virus replica-
tion on the NA function in humans (HA-mediated resistance
to NAI). Support to this concept came from the study in which
volunteers who were experimentally infected with egg-adapted
human H1N1 virus and treated with oseltamivir were moni-
tored for the emergence of drug-resistant viruses by pheno-
typic assays and sequencing of HA and NA (20). Due to its
passaging in eggs, the original virus inoculum represented a
heterogeneous mixture of receptor-binding variants differing
by amino acid substitutions in HA. In the last-day isolates from
volunteers, the ratio of the variants changed from that in the
inoculum. In the placebo group, variants with a higher affinity
for 6-linked receptors predominated over the egg-derived low-
affinity variants with typical egg adaptation mutations in HA.
By contrast, in drug-treated groups reversion of the egg-de-
rived variants to the non-egg-adapted genotype was inhibited.
These data suggested that viruses with a lower affinity for
6-linked sialic acid receptors, such as egg-adapted variants of
clinical human isolates, can be less sensitive to NAI in vivo
than original non-egg-adapted viruses. Based on these results,
it is currently believed that HA mutations decreasing virus
affinity for 6-linked sialic acids may be a primary mechanism of
the HA-mediated resistance to NA inhibitors in humans.
To study HA-mediated virus resistance to NAI in MDCK-
SIAT1 cells, we compared a clinical human virus with its three
laboratory-derived receptor binding variants, which differed
from the parent virus by single amino acid substitutions in HA.
The sensitivity of the viruses to oseltamivir carboxylate corre-
lated with their affinity for 6-linked sialic acids rather than for
3-linked sialic acids. This result suggests that neutralization
assays in MDCK-SIAT1 have the potential to adequately ac-
count for the HA-mediated resistance in humans. However,
viruses with known HA-mediated resistance in vivo have to be
tested in this assay to check whether this assumption is correct.
In summary, by stable transfection of the SIAT1 gene we
were able to make the spectrum of the sialic acid receptors in
MDCK cells more similar to that of human airway epithelium.
The sensitivity of human influenza A and B viruses to neur-
aminidase inhibitor in MDCK-SIAT1 cells was increased and
was consistent with their sensitivity in the NA enzyme assay
and with the HA receptor-binding phenotype. These features
make MDCK-SIAT1 cells a promising system for testing sen-
sitivity of influenza virus to NAI.
ACKNOWLEDGMENTS
M.M., T.M., and H.-D.K. were supported by Roche Products Ltd.,
Welwyn Garden City, United Kingdom.
We thank Robert Webster for providing clinical virus isolates, Mat-
thias Dobbelstein for his advice on cloning, and Andreas Kaufmann
for assistance with FACS analysis.
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