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Journal of Virology, February 2001, p. 1540-1546, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1540-1546.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
NSP4 Enterotoxin of Rotavirus Induces Paracellular
Leakage in Polarized Epithelial Cells
Farideh
Tafazoli,1
Carl Q.
Zeng,2
Mary K.
Estes,2
Karl-Erik
Magnusson,1 and
Lennart
Svensson3,*
Division of Medical Microbiology, Department
of Health and Environment, Linköping University,
Linköping,1 and Department of
Virology, Swedish Institute for Infectious Disease Control (SMI),
Karolinska Institute, Solna,3 Sweden, and
Division of Molecular Virology and Microbiology, Baylor College
of Medicine, Houston, Texas2
Received 27 July 2000/Accepted 7 November 2000
 |
ABSTRACT |
The nonstructural NSP4 protein of rotavirus has been described as
the first viral enterotoxin. In this study we have examined the effect
of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable
filters. Apical but not basolateral administration of NSP4 was found to
cause a reduction in the transepithelial electrical resistance,
redistribution of filamentous actin, and an increase in paracellular
passage of fluorescein isothiocyanate-dextran. Significant effects on
transepithelial electrical resistance were noted after a 20- to 30-h
incubation with 1 nmol of NSP4. Most surprisingly, the epithelium
recovered its original integrity and electrical resistance upon removal
of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented
not only development of a permeability barrier but also lateral
targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1)
protein. Taken together, these data indicate new and specific effects
of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on
polarized epithelia.
| |
TEXT |
Rotavirus is the leading cause of
infantile gastroenteritis worldwide and is associated with significant
mortality in developing countries. Despite the significant clinical
importance, the pathophysiological mechanisms by which rotavirus
induces fluid and electrolyte secretion are largely unknown. Rotavirus
infects the mature enterocytes in the mid and upper villous epithelium
of the small intestine, which ultimately leads to cell death, villous
atrophy, and diarrhea. Mechanisms that have been proposed to explain
the diarrhea include the following: malabsorption secondary to
enterocyte death (9, 16), villus ischemia (30,
34), and a toxin-like effect of the nonstructural protein (NSP4)
(2, 11, 38, 40), and the enteric nervous system plays a
key role in rotavirus fluid secretion (20).
Calcium has been shown to play an important role during rotavirus
replication and cytopathogenicity. Ca2+ homeostasis is thus
altered in rotavirus-infected cells (23, 24), and it has
been suggested that increased intracellular calcium concentrations may
be responsible for cytotoxicity and cell death (23). In
addition, it has been shown that the nonstructural NSP4 protein is
responsible for increased cytosolic Ca2+ in insect cells
and human intestinal cells and can induce diarrhea in young mice
(2, 11, 40). Furthermore, an association between increased
[Ca2+] and age-dependent Cl
secretion has
been reported (26). While the cellular mechanisms by which
NSP4 induces fluid and electrolyte changes remain unresolved, it has
been observed that NSP4 induces stimulation of inositol 1,3,5-triphosphate (IP3) production, suggesting that NSP4
mobilizes calcium through phospholipase C activation and
IP3 release (11).
Changes in intestinal permeability have previously been postulated to
contribute to intestinal secretion. Perturbations of the intestinal
epithelial barrier by enteric pathogens are not novel but the
mechanisms are probably distinct. Clostridium difficile toxins enhance permeability by disrupting actin microfilaments within
the perijunctional ring (12, 27). Vibrio
cholera (12, 48) produces specific toxins that alter
permeability by interfering with tight junctions; other pathogens,
including Salmonella (14), Shigella
(31), and enteropathogenic Escherichia coli
(EPEC) (32), can also disrupt the epithelial barrier.
Recent work with EPEC indicates that the decrease in electrical
resistance is due to disruption of epithelial tight junctions via
EPEC-induced phosphorylation of myosin light chains (49).
Tight junctions maintain the cellular polarity (apical-basolateral)
required for vectorial transport across the epithelium and provide a
barrier for passive diffusion so that the electrochemical gradient of
the epithelium can be maintained. Disruption or interference of
intestinal epithelial tight junctions may therefore contribute to
microbe-associated diarrhea. Since the distributions of both filamentous actin (F-actin) and Zona Occludens-1 (ZO-1) are altered by
certain bacteria and their toxins (17, 25, 36, 48), it is
considered that these structural changes are directly or indirectly
involved in the pathogenesis of intestinal diseases (17, 47,
48).
We (35) and others (3, 28) have successfully
employed the human intestinal Caco-2 cell line to elucidate
cytopathological effects of rotavirus. In this study, we deliberately
chose a highly polarized epithelial line, MDCK-1 cells, since a
putative effect of NSP4 could be applicable to polarized epithelia in
general. Madin-Darby canine kidney (MDCK-1) cells rapidly form a highly polarized epithelial-like monolayer on permeable supports and have been
intensively used to address cellular and physiological questions and,
more recently, also microbe-host cell interactions (4, 15, 33,
41), including rotavirus infections (35).
In this communication, we report the novel observation that the NSP4
enterotoxin of rotavirus causes a decrease in transepithelial resistance across monolayers of MDCK-1 cells and an increase in the
paracellular permeability to molecules. The data presented also show
that NSP4 prevents lateral targeting of the tight-junction-associated ZO-1 and induces disruption and/or reorganization of F-actin.
MDCK-1 cells were maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 10 mM HEPES, and 100 U of penicillin/ml.
MDCK-1 cells were grown on glass coverslips or permeable-filter
supports as described elsewhere (35, 48). Recombinant NSP4
(SA-11) and VP6 (SA-11) were produced in baculovirus-infected Sf9 cells
and were purified as previously described (39). Cells were
harvested 4 days postinfection in Hank's medium and lysed with lysis
buffer (10 mM Tris-HCl [pH 8.1]-0.1 mM EDTA-1% CHAPS
{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}). NSP4 was first semipurified by fast protein liquid chromatography using
a quaternary methylamine anion-exchange column and immunoaffinity column onto which purified rabbit immunoglobulin G against NSP4 had
been immobilized. Vp6 was purified from the medium of Sf9 cells
infected with baculovirus recombinant pAC461/Sa-11 by pelleting oligomers through a 35% sucrose cushion followed by equilibrium gradient centrifugation in CsCl (39). The baculovirus
glycoprotein gp67 was purified from lysated cells infected with
wild-type baculovirus and used as control. Protein was purified from
cells solubilized with CHAPS followed by fast protein liquid
chromatography and affinity chromatography on a concanavalin A column.
Polarized MDCK-1 cells grown on 6-mm-diameter Transwell Clear
0.4-µm-pore-size filters (0.33 cm2) (Costar) were
measured for transepithelial electrical resistance (TER) as previously
described (35, 48) by use of a Millicell-ERS resistance
apparatus (Millipore). Electrical resistance values obtained in the
absence of cells were considered background. The net resistance was
calculated by subtracting the background. The permeability of the
epithelial monolayer was assessed by measuring the rate at which
fluorescein isothiocyanate (FITC)-dextran (molecular mass, 20,000 Daltons) was transported across the epithelium. FITC-dextran was
dissolved in Krebs-Ringer glucose phosphate buffer (KRG), pH 7.3, to 20 mg/ml. Subsequently, 200 µl of this marker were added to the apical
surface of MDCK-1 monolayers and 100-µl aliquots were removed from
the basolateral compartment for a period of 46 h and placed in 2 ml of KRG for measurements. Measurements were performed using a
fluorescence spectrometer (Perkin-Elmer Ltd., Beaconsfield,
Buckinghamshire, England) with an excitation wavelength of 483 nm and
an emission wavelength of 517 nm. Monolayers were grown on permeable
filters or glass coverslips. After incubation for the desired time with
NSP4, cells were washed twice in PBS and fixed with 2.5%
paraformaldehyde for 45 min on ice, washed once in PBS, and then
incubated two times for 10 min each in 0.5-mg/ml NaBH4 to
quench free aldehyde groups. After another rinse with PBS, the cells
were permeabilized with 0.3% Triton X-100 for 7 min at room
temperature and stained with tetramethyl rhodamine isocyanate
(TRITC)-labeled phalloidin (Sigma) diluted 1:200 in PBS for 45 min at
37°C in the dark. The ZO-1 protein was detected with the primary rat
anti-ZO-1 monoclonal antibody (1520; Chemicon Int., Inc., Temecula,
Calif.) diluted 1:200 in PBS for 1 h at 37°C, then washed three
times in PBS, and then incubated for 1 h at 37°C with Alexa
488-conjugated-labeled goat anti-rat antibody (Molecular Probes,
Eugene, Oreg.). After staining and a final wash in PBS, the cells were
mounted in 4 ml of Citifluor-glycerol (Citifluor L.T.D., London, United
Kingdom), 2 g of Airvol-203 (Air Products, Utrecht, Netherlands),
and 8 ml of 0.2 M Tris-HCl (pH 8.5). Cells were examined with a 60×
immersion objective (numerical, 1.4) in a confocal laser scanning
microscope (Sarastro 2000; Molecular Dynamics, Sunnyvale, Calif.). The
514/488 nm line of the argon laser was used to excite TRITC.
Apically administered NSP4 cause a decrease in TER in MDCK-1
cells.
The permeability of polarized epithelial monolayers can be
assessed by measurement of electrical resistance across the monolayer, which measures the integrity of the tight junction and paracellular permeability. To determine whether NSP4 has any effect on the permeability on polarized epithelial cells, MDCK-1 cells were grown on
permeable supports. MDCK-1 monolayers had a TER of 
1000 to 2000 ohm/cm2 at the time of the experiments. To determine any
effect of NSP4 on the permeability, MDCK cells were treated from the
apical or basolateral compartment with a 50-µl volume containing 1 nmol (20 µM) of NSP4, VP6, or buffer followed by measuring the
electrical resistance at different times (Fig.
1A). The amount (1 nmol) was chosen
because it is capable of inducing diarrhea in mice (2). As
illustrated in Fig. 1A, apical but not basolateral administration of
NSP4 caused a time-dependent decrease in TER. A separate set of
experiments was performed to investigate whether any effect of NSP4
would occur within the first 4 h. No effect was seen (not shown),
nor were any effects with VP6 observed at any time point. The viability
of polarized MDCK-1 monolayers treated with NSP4 was examined by light
and confocal microscopy during the experimental period. By this
criterion, the cells remained viable during the experimental period.
This is in agreement with our and others' previous observations that
rotavirus induces a transepithelial leak on filter-grown polarized
epithelia before development of cythopathic effect and cell death
(28, 35).
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FIG. 1.
(a) Asymmetric effect of NSP4 on the integrity of
polarized MDCK-1 cells. NSP4 and VP6 were administered (50 µl) to the
apical (A) or the basolateral (B) surfaces of filter-grown polarized
MDCK-1 monolayers, followed by determination of TER at different time
points. TER is presented as a percent of the TER of mock-treated cells.
Mean values are presented (n = 4). (b) NSP4
demonstrates a dose-dependent effect on MDCK-1 cells. NSP4 and VP6 were
administered (50 µl) in different concentrations to the apical
surface of filter-grown MDCK-1 cells, and resistance was determined
after different time points. Mean values are presented (n = 4).
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To investigate if NSP4 causes a dose-dependent effect, MDCK-1 cells
were incubated with various amounts of NSP4, from 1 to
0.001 nmol.
Figure
1B shows that the effect of NSP4 is dose dependent,
with no
effects at amounts of 0.01 nmol or lower and a limited
effect at 0.1
nmol.
NSP4 induces paracellular passage of FITC-dextran in MDCK
cells.
To further examine the effect of NSP4 on epithelial
integrity, MDCK-1 monolayers grown on permeable filters were incubated with 1 nmol of NSP4. FITC-dextran and NSP4 were added to the apical surface at day 2 when the cells had reached an integrity of 2,000 ohm.
FITC-dextran was chosen as a paracellular marker, and flux was measured
by collecting the media from the basolateral domain at the indicated
times. As shown in Fig. 2, FITC-dextran
was transported from the apical domain to the basolateral chamber more
significantly in NSP4-treated cells than in mock-treated cells,
suggesting that NSP4 alters the tight-junction complex. Vertical
confocal sectioning revealed no sign of transcellular passage of
dextran (not shown). The kinetics of the transport corresponds well
with the kinetics of reduction in TER shown in Fig. 1 and suggests that
NSP4 alters tight-junction structure and formation.
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FIG. 2.
NSP4 (1 nmol) but not VP6 (1 nmol) increases the
paracellular flux of FITC-dextran (molecular weight, 20,000) across
polarized filter-grown MDCK-1 monolayers. Shown is the total amount of
FITC-dextran (in percent) transported from the apical to the
basolateral compartment after various time points.
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Epithelial integrity is restored by removing NSP4.
The fact
that NSP4 decreased the TER in a dose-dependent and time-dependent
manner raised a question of whether the effect would continue even
after removal of NSP4. To address this question, 1 nmol of NSP4 was
added to MDCK-1 monolayers followed by monitoring the electrical
resistance. At 23 or 50 h postadministration, when the electrical
resistance was 40 and 20% of the resistance of mock-treated cells,
respectively, NSP4 was removed, and the apical surface of the cells was
washed twice with DMEM and then incubated in DMEM for up to 96 h.
As illustrated in Fig. 3A, the TER
recovered and reached about 70% of the control level. Removal of NSP4
at 23 h (Fig. 3B) had a similar effect, except that the
regeneration of electrical resistance was slightly more efficient, most
probably due to the longer recovery time (73 h versus 46 h).
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FIG. 3.
TER is recovered after removing NSP4. (A) NSP4 (1 nmol)
was added to the apical surface of polarized MDCK-1 monolayers and then
washed out (W) with DMEM after 50 h, followed by incubation of the
cells in DMEM up to 96 h. Values are means ± 1 standard
deviation) and are presented as a percent of the resistance measured in
mock-treated cells (n = 3). (B) Recovery of TER after
removing NSP4 (1 nmol) by washing out NSP4 after 23 h
(n = 3).
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NSP4 prevents development of electrical resistance and lateral
targeting of the tight-junction-associated ZO-1 protein.
Given the
effect of NSP4 on permeability, we next investigated whether NSP4 could
prevent development of a tight polarized epithelium. MDCK-1 cells were
seeded on permeable supports for 2 days, followed by incubation with 1 nmol of NSP4, 1 nmol of VP6, or mock treatment. Figure
4 shows that coculturing of MDCK-1 cells
with 1 nmol of NSP4 significantly (day 5) prevented the development of
electrical resistance. Note that NSP4-treated cells had a slight
increase in electrical resistance from the day of administration (day
2) to day 4. This most likely reflects the incubation time required for
NSP4 to show any measurable effect on MDCK-1 cells (Fig. 1 and 2).
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FIG. 4.
NSP4 prevents development of a tight polarized
epithelium. MDCK-1 cells were cocultured with 50 µl containing 1 nmol
of NSP4 (20 µM) or VP6 from the first day of seeding the cells onto
filters. Electrical resistance measurements started on day 2 postseeding of cells on filters. Values of TER presented are means ± 1 standard deviation (n = 3). MDCK, cultivation
without protein in the medium.
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The fact that NSP4 prevented development of electrical resistance
raised the question of whether this was due to interference
with ZO-1,
a ubiquitous tight-junction-associated protein in MDCK-1
cells
(
1). To investigate if the targeting of ZO-1 to tight
junctions was affected by NSP4, MDCK-1 cells grown on a coverglass
were
incubated at days 1, 2, and 3 postseeding with 1 nmol of
NSP4 or VP6,
and the distribution of ZO-1 was analyzed by confocal
microscopy. In
contrast to mock- and VP6-treated cells, NSP4 prevented
lateral
targeting of ZO-1 and its association with tight junctions
led to a
weaker and interrupted distribution of ZO-1 (Fig.
5).
The effect was most pronounced in
cells treated for 48 h, with
NSP4 added at the time of seeding
(day 1), and was absent or weak
when NSP4 was incubated for 24 h
to already-confluent MDCK-1 monolayers
(day 3). This suggests that NSP4
interfered with the targeting
of ZO-1 to tight junctions during
biogenesis and formation of
an epithelial barrier.
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FIG. 5.
Ability of NSP4 to redistribute ZO-1 in
polarized MDCK-1 cells. Day 1, MDCK cells mixed with 1 nmol of NSP4 or
VP6 and seeded on filters for 48 h; day 2, MDCK-1 cells incubated
24 h from day 2 with NSP4 or VP6; day 3, incubation of MDCK-1
cells with NSP4 or VP6 for 24 h from day 3 postseeding on
filters.
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NSP4 alters the distribution of F-actin.
Actin filaments are a
key in maintaining cell shape and regulating tight junction
permeability, and several bacterial enterotoxins previously have been
found to alter actin microfilaments (27, 42, 47). We
therefore examined if the viral NSP4 toxin had any effect on the
distribution of F-actin in MDCK-1 cells. After a 24-h incubation of
confluent MDCK-1 monolayers with 1 nmol of NSP4, the cells were stained
with phalloidin, which binds specifically to F-actin, and examined by
confocal microscopy. Control or VP6-treated monolayers revealed a
punctate surface staining consistent with the F-actin bundles present
in the apical microvilli. As illustrated with a vertical section (Fig.
6), the apical concentration of F-actin
was much lower in MDCK-1 cells after incubation with NSP4 than in mock-
or VP6-treated cells. Colors reflect different relative concentrations
of F-actin (linear 8-bit scale = 256 levels). Figure 6 shows that
the predominant amount of F-actin is localized mainly to the apical
surface.
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FIG. 6.
NSP4 perturbs F-actin distribution in polarized MDCK-1
cells. Shown is a vertical section (x-z) of mock-treated
MDCK-1 cells (A) or MDCK-1 cells treated for 24 h with 1 nmol of
NSP4 (B) or 1 nmol of VP6 (C). F-actin was identified by TRITC-labeled
phalloidin and analyzed by confocal microscopy. The vertical sections
were scanned with a 0.35 pixel size and a pinhole setting of 50 µm.
The pseudocolors in the images represent the relative concentration
(linear 8-bit scale) of F-actin. White shows the maximum intensity
(level 256) and dark blue shows the minimum intensity (level 0).
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The mechanism of rotavirus diarrhea is not yet well understood. In
several animal species, profuse diarrhea occurs prior to
detection of
histologic changes, including villus blunting (
8,
37,
43),
and in vitro studies show that polarized intestinal
cells can withstand
infection without lysis for a long time (
19,
35). It is
therefore tempting to speculate that specific signals
elicited by
infection participate in pathophysiology. In support
of this
hypothesis, Ball et al. (
2) have shown that young mice
respond with diarrhea soon after inoculation with purified NSP4
without
significant changes of the mucosal morphology. This and
similar
observations therefore indicate that rotavirus diarrhea
somehow
involves NSP4 and that fluid secretion is not the only
direct result of
virus replication and gross morphological changes
in the
mucosa.
Polarized epithelial cells grown on permeable supports were used to
assess whether NSP4 of rotavirus had any effect on membrane
permeability. The role of increased permeability in rotavirus-induced
diarrhea remains speculative, but an association with diarrhea
has been
suggested for other diseases (
21,
22). We
(
35)
and others (
3,
28) have previously shown
that rotavirus infection
of polarized epithelial (MDCK and Caco-2)
cells leads to a paracellular
leak and F-actin alterations, and this
study extends these observations
and associates the NSP4 enterotoxin
with this effect. The time
and kinetics by which NSP4 induces a
paracellular leak are very
similar to observations of infectious
viruses in polarized epithelia
(
10,
35). FITC-dextran was
used to demonstrate NSP4-induced
flux across the epithelium, and the
kinetics paralleled the reduction
in electrical resistance. As
FITC-dextran is not cell permeable,
our present results and previous
proposal (
35) indicate that
the reduction in TER is an
effect on the paracellular
pathway.
The observation that the electrical resistance could be recovered
following removal of NSP4 indicates that apical administration
of NSP4
does not kill the cell. Instead, the kinetics suggest
that NSP4
interacts with a specific receptor on the apical surface
of MDCK-1
cells that triggers a signal transduction cascade (
11).
The decrease in TER followed the kinetics observed for native
virus
(
35) and was slower than that for certain bacteria
(
4,
7,
14,
48) but similar to that for
C. difficile toxin B
(
17).
Dong et al. (
11) have shown that exogenous treatment of
cells with NSP4 mobilizes intracellular Ca
2+ in human
intestinal cells through receptor-mediated phospholipase
C activation
and inositol-1,4,5-triphosphate production. In cells
with phospholipase
C activation, diacylglycerol is produced, which
can activate protein
kinase C. Activation of protein kinase C
has previously been reported
to participate in the regulation
of the paracellular barrier
(
6), and decreases of the electrical
resistance in MDCK-1
cells (
29) and may thus offer an attractive
explanation
for the observed decrease in TER in NSP4-treated cells.
Furthermore, it
is interesting to note that several bacterial
toxins also have specific
effects on F-actin, with a concomitant
effect on epithelial
permeability (
5,
13,
17,
18,
48).
Rotavirus has recently been shown to decrease the apical expression of
sucrase-isomaltase (
19), a brush border disaccharidase
expressed in the small intestine. A possible explanation for this
reduction could be virus-induced reorganization of the cytoskeleton
of
microvilli. In fact, rotavirus has recently been shown to perturb
the
organization of apical F-actin (
19), and this observation
is in accordance with our earlier observation that microvilli
are
affected in rotavirus-infected cells (
35). The present
study,
for the first time, associates the perturbing effect on F-actin
with the specific protein NSP4. F-actin staining was diminished
at the
apical pole of NSP4-treated cells, most likely due to depolymerization
of actin with further changes on the Zona Occludens and tight
junctions. It is interesting to note that Obert et al.
(
28)
and Dickman et al. (
10) have recently
shown that infection with
rhesus rotavirus redistributes occludin and
ZO-1. It therefore
appears that rotavirus interferes with at least two
tight-junction-associated
proteins (ZO-1, occludin), and in this study
we report that NSP4
specifically interferes with ZO-1.
Jourdan et al. (
19) have found that rotavirus perturbs the
targeting of sucrase-isomaltase to the apical surface of polarized
epithelia and proposed that rotavirus infection interferes with
targeting of this molecule to the plasma membrane. Several endogenous
proteins and rotavirus VP7 are also mistargeted in neurons infected
with rotavirus (
44-46), which further supports the
proposal that
rotavirus interferes with the secretory pathway. In this
work,
we found that rotavirus not only prevented lateral targeting of
the ZO-1 protein to tight junctions during biogenesis but showed,
more
importantly, that this novel effect could be associated with
NSP4.
In summary, our results indicate that NSP4 specifically perturbs the
paracellular pathway, reorganizes F-actin, and prevents
transport of
the ZO-1 protein to tight junctions during biogenesis
and thereby
impairs normal formation of tight junctions and a
tight polarized
epithelium.
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ACKNOWLEDGMENTS |
This study was supported by grants from the Swedish Medical
Research Council (projects 10392 [L.S.] and 6251 [K.-E.M.]),
Karolinska Institute Research Fund (L.S.), the Lions Fund (K.-E.M.),
Swedish Research Council for Engineering Sciences (K.-E.M.), King
Gustaf Vth 80-Year Fund (K.-E.M.), the National Institute of Health
(grant DK 30144 [M.K.E.]), and the Baylor-Karolinska Institute
Scientific Collaboration Program.
Special thanks to Mats Wolving for image analysis and presentations.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Virology, Swedish Institute for Infectious Disease Control (SMI), 171 82 Solna, Sweden. Phone: 46 8 457 26 96. Fax: 46 8 30 16 35. E-mail:
lensve{at}mbox.ki.se.
| |
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Journal of Virology, February 2001, p. 1540-1546, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1540-1546.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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