From the Departments of Microbiology and Immunology
and ¶ Pediatrics and the § Elizabeth B. Lamb Center for
Pediatric Research, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-2581
Received for publication, May 30, 2000, and in revised form, September 11, 2000
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ABSTRACT |
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Many serotype 3 reoviruses bind to two different
host cell molecules, sialic acid and an unidentified protein, using
discrete receptor-binding domains in viral attachment protein, The ligation of a virus particle to specific cell-surface
molecules is the primary interaction between the virus and its host, and as such it is a critical determinant of viral disease outcome and a
potential target for antiviral therapy. Studies with enveloped mammalian viruses, including members of the herpesvirus (1-3) and
retrovirus (4-6) families, have demonstrated that viral attachment is
more complex than a bimolecular interaction between a viral attachment
protein and a cellular receptor. Instead, attachment strategies
employed by these enveloped viruses involve multiple interactions
between several viral and cellular molecules, and these interactions
are often accompanied by dramatic conformational rearrangements of
viral proteins. In addition, many enveloped viruses employ an
adhesion-strengthening attachment strategy in which primary virus-cell
interactions require low affinity adhesion of the virus to common cell
surface molecules that are often carbohydrate in nature (7-9). This
initial phase of attachment is then followed by higher affinity
interactions between the virus and a secondary receptor on permissive
cells, an event that often triggers virus entry through direct membrane
fusion or receptor-mediated endocytosis (10-13).
In contrast to our developing understanding of mechanisms of enveloped
virus attachment, less is known about attachment strategies of
nonenveloped viruses, a group that includes several important human
pathogens. The mammalian reoviruses offer a useful experimental system
to dissect the contributions of discrete steps in the viral replication
cycle to pathogenesis in the infected host (14). Following oral
inoculation into newborn mice, serotype 1 (T1)1 reovirus strains spread
hematogenously to the central nervous system and replicate within
ependymal cells, resulting in hydrocephalus. In contrast, serotype 3 (T3) reovirus strains utilize neural spread pathways to gain access to
the central nervous system, where they replicate in a wide variety of
neurons and cause encephalitis (15, 16). Since reovirus contains a
segmented genome, it is possible to link pathogenic phenotypes to
individual viral genes using reassortant viruses. Using this approach,
it was determined that the mode of spread in the host (17) and cell
tropism in the central nervous system (18, 19) segregates with the
viral S1 gene segment, which encodes the viral attachment protein, The The presence of discrete RBDs in Cells, Viruses, and Antibodies--
Spinner-adapted murine L929
(L) cells were grown in either suspension or monolayer cultures in
Joklik's modified Eagle's minimal essential medium (Irvine
Scientific, Santa Ana, CA) supplemented with 5% fetal bovine serum
(Intergen, Purchase, NY), 2 mM L-glutamine, 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml
of amphotericin (Irvine Scientific). Suspension cultures of murine
erythroleukemia (MEL) cells were grown in F-12 medium (Irvine
Scientific) supplemented to contain 10% fetal bovine serum, L-glutamine, and antibiotics as for L cells. HeLa cells
were maintained in monolayer culture in Dulbecco's minimal essential
medium (Life Technologies, Inc.) supplemented to contain 10% fetal
bovine serum, L-glutamine, and antibiotics as for L cells.
Reovirus strains T1L, T3C44 (41, 42), and T3C44-MA (38) are laboratory
stocks. Viral titer (expressed as plaque-forming units (pfu)/ml) was
determined by plaque assay on L-cell monolayers (43). Purified virions were prepared by using third-passage L-cell lysate stocks of
plaque-purified reovirus as described previously (22). Viral particle
concentrations were determined by measurement of
A260 using a conversion factor of
2.1 × 1012 viral
particles/ml/A260 (44). Particle/pfu ratios of
stocks used for viral infectivity assays were ~250:1. Murine
monoclonal antibodies (mAbs) 9BG5 (G5) (45) and 5C6 (46) were purified from hybridoma culture supernatants (Cell Culture Center, Minneapolis, MN), and Fab fragments (Fabs) of each were prepared using the Immunopure Fab Purification system (Pierce) according to the
manufacturer's instructions.
Generation of Reovirus Mutants Differing in the Capacity to Bind
Sialic Acid--
Reassortant viruses were isolated by coinfecting
monolayers of L cells with T1L and either T3C44 or T3C44-MA.
Coinfections, plaque-purification, amplification of progeny viruses,
and genotyping of reassortant viruses were performed as described
previously (47). Two sequential rounds of reassortment were performed
to obtain monoreassortant viruses that retained the S1 gene segment from the T3 parental virus with all other gene segments derived from
T1L. These monoreassortant strains were designated T3/C44-SA Assessment of Virus-Sialic Acid Interactions Using Surface
Plasmon Resonance (SPR)--
Glycophorin and asialoglycophorin (1 mg/ml in PBS) (Sigma) were biotinylated by incubation with a
10-100-fold molar excess of sulfo-NHS-biotin (Pierce) at room
temperature for 2 h. Biotinylated glycophorin or asialoglycophorin
was injected at a concentration of 5-50 µg/ml in PBS across
duplicate flow cells of a BIAcore streptavidin chip at a flow
rate of 5 µl/min using a BIAcore 2000 instrument (Amersham Pharmacia
Biotech). Sensor chip flow cells were coated with 1000-2000
resonance units of each protein, followed by rinsing in running buffer
(Dulbecco's PBS (D-PBS)) to remove unbound protein. Purified virions
of strains SA Neuraminidase Treatment of Cells to Remove Sialic
Acid--
Terminal sialic acid residues were removed from cell-surface
carbohydrates by incubating 5 × 106 cells/ml at
37 °C for 1 h in 1.0 ml of D-PBS containing 40 milliunits/ml Arthrobacter ureafaciens neuraminidase (Sigma) (38). Cells
were washed in D-PBS to remove neuraminidase, resuspended in D-PBS, and
processed for binding assays.
Virus Radioligand Binding Assays--
Purified virus particles
(2-4 × 1013/ml in D-PBS) were iodinated using the
IODO-GEN two-step method (Pierce). Na125I (2.5 mCi) was
oxidized for 6 min in IODO-GEN tubes in a total volume of 220 µl of
D-PBS and then incubated with 4 ml of purified virus at room
temperature for 6 min. Virus was separated from unincorporated
125I on 10-ml dextran D-Salt columns
(Pierce) followed by dialysis against 4 liters of D-PBS. Labeled virus
was stored at 4 °C in the presence of 10 mM
NaN3, 5 mM 2-deoxyglucose, and 2 mM
NaF (Sigma). Specific activity of iodinated virus was 0.5 to 1 × 10
Radioligand binding assays were designed, and data were analyzed using
general guidelines and equations as described (52). HeLa cells were
detached from flasks by incubation in PBS plus 3 mM EDTA at
37 °C for 30 min followed by gentle pipetting. L cells were obtained
from suspension cultures. Cells were pelleted at 250 × g for 5-8 min, resuspended at 2-4 × 106/ml in D-PBS supplemented with metabolic inhibitors (10 mM NaN3, 5 mM 2-deoxyglucose, and 2 mM NaF), and incubated at 37 °C for 15-30 min to
deplete cellular ATP. This treatment abolishes receptor-mediated endocytosis in HeLa cells (53) and in L
cells.2 For experiments in
which neuraminidase-treated cells were used, 40 milliunits/ml
neuraminidase were included with metabolic inhibitors, and incubations
were extended to 1 h. Iodinated virus was diluted in D-PBS
containing metabolic inhibitors and Complete mini-EDTA-free protease
inhibitor mixture (Roche Molecular Biochemicals; final concentration
0.5× in all binding assays). Virus and cells were incubated in 1.5-ml
Eppendorf tubes at room temperature for various intervals with
continuous rotation. The viability of cells incubated in this manner
was not diminished for up to 8 h as determined by trypan blue
exclusion. At the lowest concentrations of virus used in these
experiments, equilibrium was reached within 6 h, and cell-bound
virus was stable for up to 8 h. Cell-bound virus was captured by
vacuum filtration onto Membra-fil MF MB filters (5-µm pore size)
(Whatman), followed by rinsing for 4 s with a stream of cold PBS.
Filters were air-dried, and bound virus was quantitated by liquid
scintillation counting in 4 ml of BIOSAFE II fluid (Research Products
International, Mt. Prospect, IL) using a Beckman LS6500 counter
(Beckman Instruments). Nonspecific binding was determined at several
concentrations of labeled virus by incubating duplicate samples in the
presence of 0.8-1 × 1013 unlabeled virions, the
maximum concentration of virus shown to compete virus without isotopic
dilution. Nonspecific binding was found to be a linear function
(ranging from 0.5 to 1%) of input cpm under all conditions used and
did not increase with time. For competition experiments, iodinated and
unlabeled virions were added simultaneously to cells. For experiments
assessing the effect of SLL or Fabs on virus attachment, iodinated
virions were preincubated with the indicated concentration of each
reagent at 37 °C for 30 min.
For kinetic assays, virus association (kon) and
dissociation (koff) binding constants were
derived from the observed rate of virus binding
(kobs) over time using the formula,
Assessment of Virus Growth--
Purified virions were diluted in
gelatin saline or incubated at 37 °C for 30 min in PBS alone, 50 µg/ml G5 or 5C6 Fabs, or 10 mM SLL or lactose. Tissue
culture medium was aspirated from cells (1 × 105/well
in 24-well plates), and virus inoculum (150 µl) was allowed to adsorb
to cells at room temperature. To terminate adsorption, cells were
washed with 1 ml of cold PBS, inoculum was aspirated, and 1 ml of
complete culture medium was added. Cells were incubated at 37 °C for
various intervals, followed by two cycles of freezing and thawing to
release progeny virions. Titers of infectious virus were quantitated by
plaque assay on L cells (43).
Fluorescent Focus Assays of Viral Infectivity--
Unlabeled,
purified virions were preincubated in PBS alone, 50 µg/ml G5 or 5C6
Fabs, or 10 mM SLL or lactose at 37 °C for various
intervals. To directly measure infectivity of treated virions, virus
inocula were adsorbed to confluent HeLa cell monolayers (2 × 105 cells/well) as for growth experiments. Following
incubation at 37 °C for 18 h to permit completion of a single
round of viral replication, cell monolayers were fixed with 1 ml of
methanol at Generation of Isogenic Reovirus Mutant Strains Differing in the
Capacity to Bind Sialic Acid--
To dissect the contributions of
discrete reovirus-receptor interactions to virus attachment and host
cell infection, we used reassortant genetics to construct isogenic
reovirus strains that differ only in the capacity to bind sialic acid.
T3C44 is a T3 reovirus field isolate that does not bind sialic acid.
This strain was adapted to sialic acid binding by serial passage in MEL
cells, which only support replication of sialic acid-binding strains. The Quantitation of
Since SPR measures virus-sialic acid interactions in real time, it was
possible to directly quantitate the apparent avidity, on-rate, and
off-rate of reovirus for sialic acid using nonlinear regression
analysis (Table I). When SA+
binding to glycophorin was analyzed in this manner, assuming a
reversible, 1:1 binding model (49, 51), the avidity of virus for the
glycophorin surface was 4.8 (± 1.9) × 10 Growth of SA Effect of RBD Inhibitors on Replication of Strains SA Steady State Binding of SA Kinetic Binding of SA
The derivation of kon by comparison of
kobs at various virus concentrations allows the
indirect inference of the koff of virus binding,
since koff is independent of the free virus
concentration (52). Interestingly, although SA+ displayed a higher
avidity for HeLa cells than SA Effect of Cell-Surface Sialic Acid on Cell-Attachment
Kinetics of SA Capacity of SA Defining the Role of Sialic Acid Binding in Attachment of
Infectious Reovirus Virions to Biologically Relevant
Receptors--
Since the vast majority of reovirus particles (>99%)
are not capable of productive infection in a plaque assay, it remained a formal possibility that biochemical assessments of the role of sialic
acid binding in reovirus attachment might be applicable only to
inactive particles, while infectious particles might behave differently. In addition, it seemed possible that binding to sialic acid might represent a nonproductive attachment route that is biochemically detectable but biologically irrelevant. To exclude these
possibilities, we assessed the binding rates of strains SA Temporal Coordination of Reovirus-Sialic Acid and Reovirus-Head
Receptor Interactions--
Since the capacity to bind sialic acid
enhances the association rate of strain SA+ binding to HeLa cells, we
hypothesized that sialic acid binding might serve as the initial
interaction between the virion and the cell surface. To determine
whether adhesion of reovirus to cell-surface sialic acid temporally
precedes interaction of the To dissect mechanisms by which utilization of sialic acid as a
coreceptor contributes to virus attachment and tropism, we isolated
reovirus strains that differ solely in the capacity to bind sialic
acid. Although we cannot formally exclude the existence of mutations in
these strains in gene segments other than S1, four lines of evidence
argue against this possibility. First, since T1L has been passaged in L
cells since its isolation (58) and reovirus strains are exceptionally
genetically stable in this cell line (47, 48), it is unlikely that
additional mutations were selected in other gene segments during the
few cycles of replication required to generate the monoreassortant
strains. Second, SA We found that the capacity to bind sialic acid has distinct, cell
type-dependent effects on reovirus growth that correlated with virus binding avidity and kinetics. MEL cells display an absolute
requirement for binding to sialic acid, since SA To exclude the possibility that the accelerated binding observed for
SA+ was applicable only to noninfectious particles (59), we compared
the capacity of infectious particles of SA A number of conclusions can be drawn from these results. First, the
enhanced attachment of SA+ measured in radioligand binding experiments
can be correlated with an enhanced rate of productive host cell
infection. Second, enhanced attachment mediated by sialic acid binding
is operant early in adsorption and may represent the initial attachment
event between virus and cell. Third, virus binding to sialic acid alone
is not likely to mediate efficient virus infection of HeLa cells, since
interaction between Taken together, these findings support a 1. To
determine mechanisms by which these receptor-binding events cooperate
to mediate cell attachment, we generated isogenic reovirus strains that
differ in the capacity to bind sialic acid. Strain SA+, but not SA
,
bound specifically to sialic acid on a biosensor chip with nanomolar
avidity. SA+ displayed 5-fold higher avidity for HeLa cells when
compared with SA
, although both strains recognized the same
proteinaceous receptor. Increased avidity of SA+ binding was mediated
by increased kon. Neuraminidase treatment to
remove cell-surface sialic acid decreased the
kon of SA+ to that of SA
. Increased
kon of SA+ enhanced an infectious attachment
process, since SA+ was 50-100-fold more efficient than SA
at
infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid
binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the
first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of
virus to the cell surface where access to a proteinaceous receptor is
thermodynamically favored.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (20, 21). These findings suggest that reovirus tropism and disease
outcome are determined by interactions between cell-surface receptors
and the
1 protein.
1 oligomer has two structural domains, an amino-terminal fibrous
tail, which anchors
1 at each of the icosahedral vertices of the
virion, and a compact globular head located at the carboxyl terminus
(22, 23). T3
1 recognizes at least two cellular molecules using
discrete, independently functional receptor-binding domains (RBDs).
Sequences in the
1 head domain of all T3 strains bind to an unknown
cellular molecule that is probably protein in nature. Recognition of
the
1 head receptor is critical for virus binding and infection
in vitro (24-30), and this interaction modulates tropism
and disease outcome in infected mice (31-33). Sequences in the
1
tail domain of some T3 strains confer the capacity to bind terminal
-linked sialic acid residues on glycosylated cell surface structures
(30, 34-39). Recognition of sialic acid by the
1 tail RBD can serve
as a tropism determinant for some cell types in culture (38, 40);
however, it is not clear whether binding to sialic acid is sufficient
to permit virus entry or merely facilitates
1-head receptor
interactions. The mechanisms by which sialic acid binding and
1 head
receptor binding cooperate to achieve stable, cell-specific reovirus
attachment and entry in cultured cells and in the infected host are unknown.
1 suggests that reoviruses employ
multi-step binding processes analogous to those described for some
enveloped viruses. However, attachment mechanisms used by reovirus may
be unique, since reoviruses are nonenveloped, and both RBDs exist at a
distance of a few hundred angstroms within the same viral protein.
Studies to determine mechanisms by which the tail and head RBDs of
1
cooperate to achieve stable virus-cell association, specific host cell
tropism, and modulation of disease in the infected animal have not been
possible to date, since isogenic virus strains differing only in the
capacity to utilize a distinct receptor have not been available. To
address these questions, we selected reovirus mutants that differ in
sialic acid-binding capacity, and we used reassortant genetics to place
the
1-encoding S1 genes of these strains into identical genetic
backgrounds. Using these strains, we demonstrate that reovirus
attachment is a multistep process, with primary sialic acid binding
serving as a low affinity adhesion event that accelerates virus
attachment but must be followed by
1-head receptor interactions to
permit efficient virus entry and infection.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
T3/C44-MA-SA+, abbreviated SA
and SA+, respectively, throughout this
paper. Electrophoretic mobility of monoreassortant gene segments was
verified by purifying genomic dsRNA from CsCl-banded purified virions
(5 × 1011 particles) using TriReagent (Molecular
Research Center, Cincinnati, OH). Purified gene segments were resolved
in 7% SDS-polyacrylamide gels for 550 (small class gene segments), 930 (medium class gene segments), or 1380 (large class gene segments)
mA·h at constant current, stained with EtBr, and visualized by UV
transillumination. Following reassortment, the S1 gene segments of both
strains were amplified by reverse transcription-polymerase chain
reaction using primers complementary to the 5'- and 3'-untranslated
regions (UTRs) (48), cloned into the pCR 2.1 vector, and sequenced
using T4 DNA polymerase (Sequenase 2.0, U.S. Biochemical Corp.). In
addition, the S1 gene UTRs of each strain were sequenced by direct
dsRNA sequencing of purified viral genomic RNA as described previously (37).
and SA+ were injected across the conjugated chip
surfaces at various concentrations at a flow rate of 25 µl/min.
Following virus binding, chip surfaces were regenerated with a 1-min
pulse of 1 M NaCl, 20 mM NaOH. Surfaces treated
in this manner showed no significant decrease in virus-binding capacity
for up to 30 cycles of regeneration. In some experiments, virions were
preincubated with
-sialyllactose (SLL) or lactose (Sigma) at room
temperature for a minimum of 10 min prior to injection. Since the
refractive index of concentrated virus and saccharide solutions caused
significant bulk shift effects during injection, specific binding of
virions to sialic acid was calculated by subtracting resonance units
measured on asialoglycophorin-coated reference surface from binding to
the glycophorin-coated surface prior to data analysis. No
time-dependent increase in virus binding to asialoglycophorin was detected under any conditions. Affinity constants
of strain SA+ binding to sialic acid were determined using simultaneous
kon/koff nonlinear
regression with BIAevaluation 3.0 software (Amersham Pharmacia
Biotech), assuming either a 1:1 Langmuir binding model or a bivalent
analyte binding model (49-51).
6 cpm/particle. Greater than 85% of cpm
was precipitable from iodinated virus preparations using acetone
precipitation or immunoprecipitation with anti-reovirus mAbs. Iodinated
virion preparations were used for up to 4 weeks after iodination.
Iodination of virions did not affect
1 function as assessed by
hemagglutination assay or interaction with conformationally
sensitive anti-
1 mAb G5, and no iodination of
1 was detected
following SDS-polyacrylamide gel electrophoresis analysis of labeled
reovirus virions.
and by comparing kobs for each strain at
multiple virus concentrations to exclude the contribution of
koff. This method permits the simultaneous
determination of kon and indirect inference of koff (52).
(Eq. 1)
20 °C for a minimum of 30 min. Fixed monolayers were
washed twice in PBS, blocked with 5% immunoglobin-free BSA
(Sigma-Aldrich) in PBS, and incubated at 37 °C for 30 min with
protein A-affinity-purified polyclonal rabbit anti-reovirus serum (54)
at a 1:800 dilution in PBS plus 0.5% Triton X-100. Monolayers were
washed twice in PBS plus 0.5% Triton X-100 and incubated at 37 °C
for 30 min with a 1:1000 dilution of anti-rabbit goat Ig conjugated
with Alexa488 (Molecular Probes, Inc., Eugene, OR). Monolayers were
washed twice, and infected cells were visualized by indirect
immunofluorescence. Infected cells were identified by the presence of
intense cytoplasmic fluorescence that was excluded from the nucleus. No
background stain was noted on uninfected control monolayers. Reovirus
antigen-positive cells were quantitated by counting fluorescent cells
in three random fields of view per well at 100-400 × magnification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-encoding S1 gene segment of the MEL-adapted variant (T3C44-MA) was sequenced and was found to possess a single coding mutation (Leu204
Pro) in a region of the
1 tail previously
implicated in carbohydrate binding (38). However, since we thought it
possible that additional mutations might have arisen in other gene
segments during serial passage in MEL cells, we used reassortant
genetics to place the S1 gene segments of T3C44 and T3C44-MA into the
genetic background of T1L, a T1 strain that does not bind sialic acid
(34, 38). Following two successive rounds of coinfection and genetic
screening, two viruses were isolated that contained the S1 gene of
either T3C44 (strain SA
) or T3C44-MA (strain SA+), and all other gene segments from T1L (Fig. 1). Following
reassortment, the S1 genes of both monoreassortant strains were
sequenced and were found to contain no additional mutations (data not
shown).
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Fig. 1.
Generation of reovirus strains that differ
only in the capacity to bind virus coreceptor sialic acid.
Electrophoretic mobility of gene segments of parental viruses
(T1L, T3C44, and T3C44-MA) and
monoreassortant viruses (SA and SA+) is shown.
Purified genomic dsRNA from each virus strain (5 × 1011 particles) was separated in three SDS-polyacrylamide
gels to maximize resolution, stained with EtBr, and visualized by UV
transillumination. The S1 gene segments are indicated by
arrows, with large (L), medium (M),
and small (S) class genes designated.
1-Sialic Acid Interactions Using an SPR
Biosensor--
To obtain a quantitative assessment of the avidity of
SA
and SA+ for sialic acid, we utilized SPR to measure interactions between virions and sialic acid residues in real time in the absence of
molecular labeling. The erythrocyte glycoprotein bound by T3 strains in
hemagglutination assays is glycophorin A (55), and treatment of
glycophorin with neuraminidase to remove terminal sialic acid residues
abrogates virus binding to this protein (37, 39). We therefore used
biotin/streptavidin chemistry to attach glycophorin and
asialoglycophorin to an SPR detector chip and assessed the capacity of
SA
and SA+ to bind to these surfaces. The asialoglycophorin-coated
chip served as a reference surface to measure nonsialic
acid-dependent interactions between virus and either
glycophorin or the chip surface (49, 50). We found that SA
was
incapable of binding to either glycophorin or the asialoglycophorin
surface, while SA+ bound saturably and specifically to glycophorin
(Fig. 2A) but not to
asialoglycophorin (data not shown). To confirm that binding of SA+ to
glycophorin was sialic acid-mediated, we tested the capacity of
SLL, a soluble trisaccharide that mimics the
-linked sialic
acid residues of cellular oligosaccharide chains, to inhibit virus
binding to the glycophorin-coated biosensor surface (Fig.
2B). SLL inhibited binding of SA+ to glycophorin in a
dose-dependent manner, displaying an IC50 of
~500 µM and achieving complete inhibition at 4 mM. Preincubation of virions with 4 mM lactose
had only a modest inhibitory effect on SA+ binding to glycophorin,
indicating that the effect of SLL was mediated specifically by the
sialic acid residue (Fig. 2B).
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Fig. 2.
Quantitation of sialic acid binding avidity
of reovirus strains SA and SA+ using SPR. Purified virions at
the concentrations shown were injected over a biosensor surface using a
BIAcore 2000. Sensor surfaces were coated with either glycophorin or
asialoglycophorin as control. All binding data were adjusted to
subtract refractive index bulk shifts and nonspecific binding to the
asialoglycophorin surface, which represented less that 10% of specific
binding to glycophorin using all conditions tested. A,
kinetic binding of strains SA
and SA+ to sialic acid. Strain SA+ at
4 × 1013 parts/ml (a), 3 × 1013 parts/ml (b), 2 × 1013
parts/ml (c), 1 × 1013 parts/ml
(d), 5 × 1012 parts/ml (e),
2.5 × 1012 parts/ml (f), or SA
at 4 × 1013 parts/ml (g) was injected across the
biosensor surface, and binding was measured. Virus and buffer injection
times are indicated. B, the capacity of soluble SLL to
inhibit binding of strain SA+ to sialic acid was assessed by
preincubation of virions with PBS (a) or with SLL at 250 µM (c), 500 µM (d), 1 mM (e), 2 mM (f), or 4 mM (g) prior to injection over the biosensor
surface. Parallel incubations of virions with 4 mM lactose
were used as control (b). All biosensor experiments were
performed at least three separate times in duplicate using
independently prepared biosensor chips. Shown are representative
experiments.
9 M. In addition, analysis of
SA+ binding to sialic acid suggests that this interaction is likely to
be multivalent, since nonlinear regression using bivalent binding
assumptions (49) more closely approximated the binding of SA+ to sialic
acid (
2 = 0.23 ± 0.04 versus 2.3 ± 1.5 for 1:1 binding). These results indicate that a single mutation
in the
1 tail can confer high affinity binding of reovirus to sialic
acid.
Equilibrium and kinetic binding constants
and SA+ in L cells, MEL Cells, and HeLa
Cells--
To determine whether the sialic acid-binding phenotypes of
strains SA+ and SA
confer biological differences in viral tropism for
discrete cell types, we assessed the yields of these strains after
growth in L cells, MEL cells, and HeLa cells (Fig.
3). Murine L fibroblasts are used
routinely to cultivate reovirus, and these cells do not require sialic
acid binding for productive infection (34-36). Both SA
and SA+ grew
efficiently in L cells, generating between 100 and 10,000 progeny pfu
per input pfu. In contrast, only strain SA+ was capable of growth in
MEL cells, which display an absolute requirement for sialic acid
binding for attachment and infection by reovirus (38, 40). HeLa cells
displayed a phenotype intermediate to that of L cells and MEL cells;
both strains grew in these cells, but strain SA+ displayed an
80-100-fold increase in viral yield in comparison with SA
(Fig. 3).
Thus, the capacity to bind sialic acid enhances growth of reovirus in HeLa cells but is not absolutely required for infection.
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Fig. 3.
Growth of SA and SA+ in L cells, MEL cells,
and HeLa cells. The capacity of strains SA
and SA+ to infect L
cells, MEL cells, and HeLa cells was determined by adsorption of cells
(1 × 105) with virus at an MOI of 1 pfu/cell,
followed by incubation at 37 °C for 24 h. Virus present at 0 and 24 h was quantitated by plaque assay using L cell monolayers.
Viral growth, expressed as viral yield, is equal to the
log10 of pfu/ml at 24 h divided by pfu/ml at 0 h.
Viral yields were assessed in triplicate experiments. Error
bars represent S.D.
and
SA+--
To determine the relevance of each RBD-receptor interaction
during a complete replication cycle, the effect of RBD inhibitors on
virus replication in HeLa cells was assessed (Fig.
4). Strains SA
and SA+ were incubated
with SLL, lactose, G5 Fabs, or 5C6 Fabs prior to adsorption to HeLa
cells. The mAb G5 binds to the
1 head domain and neutralizes virus
infection by blocking binding to the cellular receptor for the head
(39, 45), and 5C6 is an isotype-matched control antibody (46). G5 Fabs
reduced growth of both SA
and SA+ ~20-fold. SLL had no effect on
growth of SA
but decreased growth of SA+ ~10-fold. This effect was
specific, since lactose did not decrease SA+ growth. A combination of
G5 Fabs and SLL almost completely inhibited growth of SA+. These data
indicate that the
1 head RBD of SA
alone mediates infectivity of
this strain, while the
1 sialic acid and head RBDs of strain SA+
cooperate to promote enhanced viral replication in HeLa cells.
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Fig. 4.
Role of 1 RBDs in
viral growth in HeLa cells. Purified virions of SA
and SA+ were
preincubated at 37 °C for 30 min with PBS alone, SLL (10 mM), lactose (LACT) (10 mM), G5 Fabs
(50 µg/ml), 5C6 Fabs (50 µg/ml), or G5 Fabs and SLL combined and
adsorbed to HeLa cells (1 × 105) in monolayer culture
at an MOI of 1 pfu/cell. After a 24-h incubation at 37 °C, progeny
virus was released by freezing and thawing cells and titrated by plaque
assay on L cells. Viral growth, expressed as viral yield, is equal to
the log10 of pfu/ml at 24 h divided by pfu/ml at
0 h. Viral yields were assessed in triplicate experiments.
Error bars represent S.D.
and SA+ to L Cells and HeLa
Cells--
To determine the mechanisms by which sialic acid binding
enhances reovirus infection of HeLa cells, we performed radioligand binding studies using 125I-labeled SA
and SA+ virions
incubated with intact L cells and HeLa cells. Cells were ATP-depleted
to inhibit endocytic processes (53) and incubated with increasing
concentrations of iodinated virions until equilibrium was reached.
Binding of SA
and SA+ to both L cells and HeLa cells was saturable
and specific, with SA+ capable of achieving higher maximal binding and
approaching saturation at lower virus concentrations than SA
on both
cell types (Fig. 5, A and
C). However, the difference between maximal binding levels
of SA
and SA+ was greater on HeLa cells than L cells. When steady
state binding results were analyzed using a Scatchard transformation
(57), both strains demonstrated linear plots on both cell types (Fig.
5, B and D). Therefore, at concentrations of
virus used in these experiments, these viruses recognize
thermodynamically uniform receptor populations at equilibrium. The
avidity (expressed as apparent KD) of SA
for L
cells was 3.3 × 10
11 M,
(Table I), whereas the avidity of SA+ to L cells was only slightly
higher, with a KD of 2.5 × 10
11 M. The nearly equivalent
avidities of SA
and SA+ for L cells are consistent with the capacity
of these strains to replicate with equal efficiency in this cell type
(Fig. 3). In contrast, the avidity of SA
for HeLa cells was 2.5 × 10
10 M, approximately 8-fold
lower than that observed on L cells. SA+ avidity for HeLa cells was
significantly higher than SA
, with a KD of 5 × 10
11 M. Thus, the capacity to
bind sialic acid increases the avidity of reovirus for HeLa cells by
5-fold but has little effect on the avidity of either strain for L
cells. The enhanced avidity of SA+ for HeLa cells correlates with the
capacity of this strain to replicate more efficiently than SA
in this
cell type and suggests that the thermodynamics of virus-cell
interaction can be modulated by sialic acid binding in a manner that
enhances virus infection in some types of cells.
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Fig. 5.
Equilibrium binding of SA and SA+ to L
cells and HeLa Cells. A, saturation binding isotherms
of SA
and SA+ binding to L cells. Purified virions of SA
and SA+
were radioiodinated and incubated at indicated concentrations at room
temperature for 6 h with 1 × 106 cells in a
total volume of 1 ml of D-PBS. Cell-associated virus was separated from
free virus using vacuum filtration and quantitated by liquid
scintillation. Nonspecific binding, defined by parallel incubations in
the presence of 0.8-1 × 1013 particles of unlabeled
homologous virus, was subtracted from each data point. Specific virus
binding is presented as a function of increasing concentration of
radiolabeled virus. B, Scatchard transformation of
equilibrium binding data on L cells. Concentrations of free virus were
directly determined by scintillation counting of 25-µl aliquots taken
from several samples in A prior to vacuum filtration.
Apparent avidity of each strain for L cells is expressed as
KD. C, saturation binding isotherms of
SA
and SA+ binding to HeLa cells. Binding reactions were performed
and quantitated as for L cells. D, Scatchard transformation
of equilibrium binding data on HeLa cells. Apparent avidity of each
strain for HeLa cells is expressed as KD.
Experiments were performed in duplicate, and average specific binding
is shown. Error bars represent the range of data
obtained.
and SA+ to HeLa Cells--
To determine
whether sialic acid binding affects the association
(kon) or dissociation
(koff) rate of reovirus binding, we performed
kinetic binding assays with SA
and SA+ on HeLa cells (Fig.
6). During the association phase, SA+
displayed more rapid binding and a higher final equilibrium level than
SA
(Fig. 6A). When observed association rates
(kobs) were assessed at various virus
concentrations (Fig. 6, B and C) during the first
60 min of incubation, we found that the binding rate of each strain was proportional to the input concentration of virus, but that SA+ bound
more rapidly to the HeLa cell surface than SA
. Transformation of
these data to eliminate the contribution of koff
demonstrated that SA
displayed a kon of 6 × 106 min
1
M
1, while the
kon of SA+ was approximately 16-fold more rapid,
at 1 × 108 min
1
M
1 (Fig. 6C, Table I).
Thus, sialic acid binding increases the avidity of reovirus for HeLa
cells by accelerating the on-rate of virus-cell interaction.
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Fig. 6.
Kinetics of attachment of SA and SA+ to
HeLa cells. Purified, radiolabeled virions of each strain were
incubated with 1 × 106 cells for the times shown.
Binding reactions, separation of bound from free virus, and
quantitation of specific binding were performed using conditions
identical to those used for the equilibrium binding studies shown
in Fig. 5. A, association of strains SA
and SA+ with
HeLa cells. Radiolabeled virions (3 × 1011
particles) of each strain were incubated with cells, and bound virus
was quantitated at the times indicated following vacuum filtration.
Nonspecific binding was calculated from parallel incubations in the
presence of excess unlabeled virus. Nonspecific binding did not
increase with time and is subtracted from the data shown. B
and C, data used for determination of association
(kon) and dissociation
(koff) rate constants of strains SA
and SA+ on
HeLa cells. B, cell-associated virus was quantitated
following incubation of virus at the indicated concentrations for the
times shown, and untransformed data were plotted as a function of time.
C, to determine kobs for each
concentration of virus used, data from B were replotted as
ln([virus bound at equilibrium]/[virus bound at equilibrium]
[virus bound at time point]) (52). Linear regression analysis was
used to determine kobs, equal to the slope of
each binding curve. The kobs values from
C were plotted as a function of virus concentration to
calculate kon and koff in
Table I. Experiments were performed in duplicate, and average specific
binding is shown. Error bars represent the range
of data obtained.
under equilibrium conditions, the
initial koff for SA+ was 15-fold more rapid than
that of SA
. This koff would result in an
initial KD of SA+ for HeLa cells of 6.8 × 10
10 M, ~14-fold less avid than
that measured at equilibrium. In contrast, the kinetic
KD of SA
was 7.5 × 10
10 M, similar to that obtained
under equilibrium conditions (Table I). These results suggest that SA+,
but not SA
, undergoes a time-dependent increase in its
avidity for HeLa cells, likely mediated by a decrease in
koff as binding approaches equilibrium.
and SA+--
To confirm that the enhanced attachment
kinetics of SA+ are dependent on cell-surface sialic acid, we
determined the effect of neuraminidase treatment of cells on attachment
rates for each virus strain (Fig. 7).
Preincubation of HeLa cells with neuraminidase did not significantly
alter the avidity of SA
for these cells (Fig. 7A, Table
I). In contrast, the binding avidity of SA+ on neuraminidase-treated
HeLa cells was reduced to precisely that of SA
(Fig. 7A,
Table I). The decrease in affinity of SA+ for neuraminidase-treated
HeLa cells was due to a decreased rate of association of this strain
(Fig. 7B). Consistent with the hypothesis that accelerated
adsorption of SA+ mediates enhanced infectivity on HeLa cells, we also
find that neuraminidase treatment of HeLa cells reduces viral yield of
SA+ to that of SA
(data not shown). These data indicate that
cell-surface sialic acid is required to enhance the attachment rate of
SA+, but that the head receptor bound by these strains does not require
sialylation for its function in virus binding.
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Fig. 7.
Role of cell surface sialic acid in binding
of SA and SA+ to HeLa cells. HeLa cells (1 × 106) were treated with A. ureafaciens
neuraminidase to remove cell surface sialic acid prior to incubation
with radiolabeled virions of each strain at the concentrations
indicated. A, Scatchard transformation of specific virus
binding to neuraminidase-treated cells at equilibrium. Binding
reactions were performed in duplicate and quantitated as in Fig. 5.
B, kinetic binding of SA
and SA+ to mock- or
neuraminidase-treated HeLa cells. Radiolabeled virions (4 × 1011) were incubated with 1 × 106 cells.
Cell-associated virus was quantitated at the times shown. Nonspecific
binding is subtracted from all data points. The experiments were
performed in duplicate, and average specific binding is shown.
Error bars represent the range of data
obtained.
and SA+ to Compete for Cell-Surface
Receptors--
To determine whether the mutation in the
1 tail that
confers sialic acid binding to strain SA+ also alters the nature of the
interaction between the
1 head and its receptor, we tested the
capacity of each strain to compete the binding of the heterologous strain to HeLa cells (Fig. 8). Strain
SA
and SA+ competed their own capacity to bind HeLa cells in a
dose-dependent manner, with a 50-fold excess of unlabeled
virus being sufficient to reduce binding of labeled virus to the level
of background (Fig. 8, A and B). Unlabeled SA+
efficiently competed binding of strain SA
, with dramatic inhibition
of SA
attachment observed in the presence of a 5-fold molar excess of
SA+ (Fig. 8A). The enhanced capacity of SA+ to compete
binding of SA
is likely due to the accelerated kon of SA+ (Fig. 6, Table I), which would enable
it to saturate cell-surface receptors more rapidly than SA
. In
contrast, even a 100-fold excess SA
had minimal effect on the binding
of SA+, which suggests that strain SA+ has acquired the capacity to
bind an additional receptor molecule, presumably sialic acid (Fig. 8B). To test this hypothesis, HeLa cells were treated with
neuraminidase to remove sialic acid, and competition profiles of each
strain were repeated (Fig. 8, C and D). In the
absence of cell surface sialic acid, both strains bound equivalently to
HeLa cells and were capable of competing the attachment of the
heterologous strains with identical efficiency. These results indicate
that the head receptor bound by strains SA
and SA+ is identical and
suggest that the capacity to bind sialic acid confers a competitive
advantage in the capacity of strain SA+ to bind to this receptor.
Furthermore, these data suggest that SA+ can associate with the HeLa
cell surface in a stable manner via binding to sialic acid in the
absence of
1-head receptor ligation.
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Fig. 8.
Competition between SA and SA+ for binding
to HeLa cells (A and B) and
neuraminidase-treated HeLa cells (C and
D). Radiolabeled virions (8 × 1010 particles) of SA
and SA+ were incubated with 1 × 106 cells in a total volume of 1 ml in the presence of
increasing concentrations of unlabeled homologous or heterologous
strain. Cell-associated virus was quantitated after 6 h of
incubation, and nonspecific binding was subtracted from each data
point. A, competition of 125I-SA
binding by
unlabeled SA
(white bars) or SA+
(black bars) at the indicated concentrations.
B, competition of 125I-SA+ binding by unlabeled
SA
(white bars) or SA+ (black
bars) at the indicated concentrations. C and
D, prior to binding reactions, HeLa cells were treated with
A. ureafaciens neuraminidase to remove cell surface sialic
acid. Final volume for virus binding reactions was 1.5 ml.
C, competition of 125I-SA
binding by unlabeled
SA
(white bars) or SA+ (black
bars) at the indicated concentrations on
neuraminidase-treated HeLa cells. D, competition of
125I-SA+ binding by unlabeled SA
(white
bars) or SA+ (black bars) at the
indicated concentrations on neuraminidase-treated HeLa cells.
Experiments were performed in triplicate, and average specific binding
is shown. Error bars indicate S.D.
and SA+
using a fluorescent focus assay for attachment of infectious particles
to individual cells in the presence of specific inhibitors of each
1
RBD (Fig. 9). Purified virions of SA
and SA+ were titrated on L cells, and equal numbers of pfu were
preincubated with either SLL, lactose, G5 Fabs, or 5C6 Fabs for 30 min.
Following incubation with these RBD inhibitors, virus was adsorbed to
HeLa cell monolayers for increasing periods of time, unbound virus was
removed by thorough washing, and cells were incubated at 37 °C for
18 h to permit viral entry and a single round of replication. Infected cells were visualized by immunofluorescence. Both virus strains displayed a linear, time-dependent increase in
numbers of infected cells (Fig. 9, A and B).
However, infection rates of SA+ were dramatically accelerated in
comparison with SA
, with SA+ displaying 50-100-fold more fluorescent
focus units (FFU) than SA
at each time point (Fig. 9, A
and B). Differences in the capacity of SA
and SA+ to
generate FFU were not inherent to the assay, since these strains
produce FFU with equivalent kinetics on L cells (data not shown).
Preincubation with SLL significantly decreased the attachment rate of
SA+ but not SA
, indicating that the enhanced rate of infection
displayed by SA+ was dependent on virus-sialic acid interactions (Fig.
9B). Neutralizing mAb G5 potently and specifically inhibited
infection with either SA
or SA+ (Fig. 9, A and
B). These data indicate that the attachment kinetics
demonstrated using purified viral particles parallel those of
infectious particles and suggest that sialic acid binding enhances a
productive binding pathway for reovirus virions.
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Fig. 9.
Role of 1 RBDs in
adsorption of infectious virus particles. Purified, unlabeled
virions of SA
(A) or SA+ (B) were preincubated
at 37 °C for 30 min with PBS, SLL (10 mM), lactose (10 mM), G5 Fabs (50 µg/ml), or 5C6 Fabs (50 µg/ml) and
adsorbed to HeLa cells (2 × 105) in monolayer culture
at an MOI of 10 pfu/cell at room temperature for the times shown. After
an 18-h incubation at 37 °C to permit virus infection, infected
cells were detected by indirect immunofluorescence using polyclonal
rabbit anti-reovirus serum and anti-rabbit Alexa488. Data are expressed
as FFU/well. Experiments were performed in duplicate, and average FFU
are shown. Error bars represent the range of data
obtained. Note that different scales are used for the y axes
of A and B. The lactose data point in
B is obscured by the 5C6 data point and does not differ
significantly from the 90-min control sample.
1 head with its receptor, we compared
the capacity of SLL and G5 to inhibit infection of SA+ when added at
various times during the adsorption phase (Fig.
10). SLL substantially (~90%)
inhibited viral infection by SA+ when added at either 30 or 0 min prior
to adsorption. However, SLL exhibited a time-dependent decrease in its capacity to inhibit attachment of SA+ between 0 and 20 min of adsorption, with almost all inhibitory effect lost by 30 min of
adsorption. In contrast, G5 Fabs inhibited viral infection by >75%
when added at any time prior to or during adsorption (up to 45 min).
These findings suggest that the enhancement of infectivity conferred by
the capacity to bind sialic acid is mediated by adhesion of virus to
cell-surface sialic acid as a primary step, but that this interaction
is followed by transition to a sialic acid-independent interaction
mediated by the
1 head.
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Fig. 10.
Temporal coordination of
1 RBD utilization during reovirus attachment.
Purified, unlabeled virions of SA+ were adsorbed to HeLa cells (2 × 105) in monolayer culture at an MOI of 10 pfu/cell at
room temperature for the times shown. At times shown prior to or during
adsorption, SLL (10 mM) or G5 Fabs (50 µg/ml) was added
to the virus inoculum and rapidly mixed. After a 60-min adsorption,
unbound virus was aspirated in 1 ml of ice-cold PBS, prewarmed tissue
culture medium was added, and cells were incubated at 37 °C for
18 h. Infected cells were detected by indirect immunofluorescence
using polyclonal rabbit anti-reovirus serum and anti-rabbit Alexa488.
The percentage of neutralization was calculated by dividing the number
of FFU present at each time point by FFU present after 60 min of
adsorption in the absence of RBD inhibitor (see Fig. 9). Experiments
were performed in duplicate, and the average percentage of
neutralization is shown. Error bars represent the
range of data obtained.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and SA+ demonstrate cell-specific tropism
previously attributed to the S1 gene segments of their respective T3
parent strains (38). Third, the same cellular molecule is bound by the
1 head of SA
and SA+, since SA+ fully competes binding of SA
on
HeLa cells, and both viruses cross-compete on neuraminidase-treated cells. Finally, differences in binding, virus-induced signaling and
apoptosis, and replication between SA
and SA+ in HeLa cells are
mediated solely by binding to sialic acid (this
report).3 Thus, SA
and SA+
represent a site-directed mutant pair that differs genetically with
respect to a single amino acid, Leu204 (SA
)
Pro204 (SA+), in the
1 tail domain and phenotypically in
the capacity to bind sialic acid as a viral coreceptor. These strains
represent the first reovirus mutants to be isolated that differ solely
in the capacity to bind a known reovirus receptor.
did not replicate in
these cells. In contrast, L cells display no requirement for sialic
acid binding, since both SA
and SA+ grew efficiently in these cells.
Consistent with this result, we found that the steady-state avidities
of these strains for L cells were equivalent. An intermediate
requirement for binding to sialic acid was found for HeLa cells.
Although both strains were capable of infecting HeLa cells, SA+
produced greater yields than SA
. At equilibrium, the avidity of SA+
for HeLa cells was 5-fold higher than that of SA
, and this increased
avidity was attributable to an accelerated kon
of SA+. Enhanced steady-state and kinetic binding of SA+ to HeLa cells
required cell surface sialic acid, since the avidity and association
rate of SA+ decreased to that of SA
when assessed using
neuraminidase-treated cells. In contrast, although the
kon of SA+ binding to L cells was modestly
enhanced relative to SA
, the kon of SA
binding to L cells was as rapid as observed for SA+ binding to HeLa
cells (data not shown), suggesting that this kon
represents a kinetic threshold necessary for efficient viral adsorption
to cultured cells. It is important to note that given the complex
nature of the interactions between a multivalent viral ligand and the
cell surface, binding constants derived in this study for SA
and SA+
do not represent absolute biophysical properties of any single
ligand-receptor interaction occurring during the process of virus
adsorption. Instead, analysis of viral binding using the assumptions of
mass action enables the comparison of the relative avidity and kinetics
of SA
and SA+ binding to cells.
and SA+ to bind to HeLa
cells using a fluorescent focus assay that directly detects infected
cells. We found that SA+ was 50-100-fold more efficient than SA
at
establishing a biologically productive binding event. Enhanced kinetics
of SA+ infectivity were mediated at the attachment step and were
dependent on virus binding to cell-surface sialic acid, since
preincubation of virus with SLL dramatically reduced the efficiency of
SA+ infection. The capacity of SLL to inhibit infectious binding of SA+
was most potent when virus was preincubated with this compound, and
inhibition decreased rapidly over the first 30 min of adsorption to the
cell surface. In contrast, the capacity of a neutralizing antibody to
the
1 head to inhibit infectious attachment remained high throughout
the course of the adsorption phase.
1 and the head receptor continues to play a
critical function after the sialic acid-mediated adsorption phase is
complete. Finally, reovirus attachment is likely to be a temporally
regulated process, since an early, sialic acid-dependent
phase can be separated from a later, sialic acid-independent phase.
This multiphasic nature of reovirus adsorption is consistent with
binding constants for SA+ on HeLa cells, in which the initial
KD of SA+ as determined kinetically was
characterized by a rapid koff and was 14-fold
weaker than the saturation avidity but transitioned at equilibrium to a
higher avidity binding state.
1 head receptor-dominant
adhesion-strengthening model of reovirus attachment (Fig. 11). For sialic acid-binding reovirus
strains, the initial interaction between the viral particle and the
host cell is likely to be mediated by cell-surface sialic acid. This
interaction may involve multivalent virion-sialic acid interactions and
has an apparent avidity of ~10
9
M as measured on a biosensor surface. By virtue of its
rapid association rate, this interaction adheres the virion to the cell surface, enabling it to diffuse laterally until it interacts with the
1 head receptor molecule. This secondary interaction is the only
binding event available to strains that do not bind sialic acid and may
be necessary and sufficient for virus endocytosis. This attachment
strategy is similar to that proposed for the
-herpesviruses, where
primary virus-cell interactions occur between viral glycoproteins and
heparin sulfate, but virus penetration is facilitated by binding to one
of a family of herpesvirus entry mediators (1, 2, 13, 60-62).
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Fig. 11.
A multistep model for reovirus
attachment. Evidence presented in this report supports a model in
which the primary interaction between a reovirus particle and the host
cell is mediated by rapid but low affinity adhesion of virus to
cell-surface sialic acid (SA) via residues in the fibrous
tail of 1. On permissive cells, this is followed temporally by
transition to a higher affinity interaction between the
1 head and
an unidentified cell surface head receptor (HR). This
secondary interaction may trigger virus internalization and initiation
of infection.
Multiple mechanisms may explain the enhanced kinetics of 1-head
receptor interaction that are observed in the presence of sialic acid
binding. The accelerated attachment rate may simply be due to an
increase in relative concentrations of virus and head receptor that
occurs when the virus adheres to the cell surface. Alternatively, the
virus binding site on the
1 head receptor may be proximal to the
cell surface or otherwise sterically inaccessible to soluble virus. In
this scenario, virus binding to sialic acid may serve to insert the
1 head into the glycocalyx to permit interaction between this
receptor and
1 (63, 64). Finally,
1-sialic acid interactions may
induce a conformational change in
1 that places the head RBD in a
state that displays higher affinity for its receptor, in a mechanism
analogous to CD4-induced conformational changes that expose chemokine
receptor-binding residues on HIV gp120 (4-6), although a
1
conformational change alone is not sufficient to enhance attachment,
since incubation with SLL inhibits rather than enhances SA+ binding.
Discriminating between these models will require identification and
characterization of the cellular molecule bound by the
1 head.
In addition to accelerating virus attachment through adhesion to the
cell surface, the capacity to bind sialic acid may alter the biological
outcome of reovirus infection. For example, sialic acid-binding
reovirus strains may function as lectins, cross-linking sialylated
cellular molecules and perturbing intracellular signaling pathways (65,
66). Alternatively, sialic acid binding may enhance the capacity of
reovirus particles to induce cross-linking or activation of the 1
head receptor, an event that may have downstream effects, depending on
the nature of this molecule. Strain SA+ induces NF-
B activation in
infected cells, thereby triggering apoptotic cell death (67,
68).3 Strain SA
neither activates NF-
B nor causes
apoptosis, even at doses that result in infection of all cells in a
culture.3 These results support the hypothesis that binding
to sialic acid may dramatically alter steps in virus-cell interaction
following attachment and entry. Accordingly, although we favor a model
whereby sialic acid binding enhances reovirus infection by accelerating adsorption (Fig. 11), our data are also consistent with sialic acid-mediated signaling events enhancing yield of SA+ in HeLa cells at
a post-attachment step by altering the metabolic state of the host cell.
One implication of the adhesion-strengthening model for reovirus attachment proposed here is that the relative expression levels of sialic acid and head receptor on a given cell type may determine the types of virus strains that can infect these cells. On cells that express high levels of head receptor, sialic acid may be dispensable for efficient infection; L cells may represent such a cell type. Conversely, on cells that express low levels of head receptor, sialic acid binding may be an absolute requirement for infectivity, as is the case for MEL cells. At intermediate levels of head receptor, sialylated cell-surface molecules may significantly enhance the efficiency of viral attachment, like the situation observed for HeLa cells. Finally, on cells that do not express the head receptor, binding to sialic acid may represent a "dead end" binding event that does not support viral infection. These attachment scenarios may have varying degrees of importance during discrete phases of replication in the infected animal.
Despite the fact that neuroinvasion and neurovirulence phenotypes of T3
reovirus strains have been genetically mapped to the 1-encoding S1
gene, there exists little understanding of which
1-receptor
interactions dictate the exquisite neural tropism of these viruses.
Since all detailed studies of T3 pathogenesis have used sialic
acid-binding strains, the role of this receptor in neural tropism and
disease is unknown. Interestingly, a sialic acid-binding T3 strain has
been shown to cause apoptosis in the neonatal central nervous system
(69) and heart (70), suggesting that binding to this carbohydrate may
induce signaling alterations in the host as it does in cultured cells.
The enhanced kon of SA+ may be particularly
important in permitting spread through rapidly moving body fluid such
as blood or lymph (71), where adhesion to endothelial cells might
permit virus to concentrate at the cell surface in a manner analogous
to selectin-mediated lymphocyte rolling (56). In addition, results from
the current study suggest that sialic acid binding is critical for
infectivity of cell types that express the
1 head receptor at low
levels, which predicts that SA+ would display expanded tissue tropism in the host relative to SA
. Future studies using these reovirus strains that differ solely in the capacity to bind sialic acid will
enable us to dissect the mechanisms by which interaction of a virus
with a common cell-surface carbohydrate contributes to virus-induced
cell death and disease in the infected host.
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ACKNOWLEDGEMENTS |
---|
We thank Ray Mernaugh and Ergang Shi of the Molecular Recognition Unit, Vanderbilt Cancer Center Core Facility, for expert assistance in designing and interpreting biosensor experiments. We acknowledge the National Cell Culture Center for purification of monoclonal antibodies.
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FOOTNOTES |
---|
* This work was supported by Public Health Service award AI38296 from the NIAID, National Institutes of Health (NIH) and the National Science Foundation (to E. S. B.), the Vanderbilt University Research Council (to E. S. B. and J. C. F.), and the Elizabeth B. Lamb Center for Pediatric Research. Additional support was provided by Public Health Service awards CA68485 (to the Vanderbilt Cancer Center), DK20593 (to the Vanderbilt Diabetes Research and Training Center), and NCI, NIH, Grant T32 CA09385 (to J. C. F).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Lamb Center for
Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-9943; Fax: 615-343-9723; E-mail: terry.dermody@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M004680200
2 E. S. Barton and T. S. Dermody, unpublished observation.
3 J. L. Connolly, E. S. Barton, and T. S. Dermody, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
T1 and T3, serotype
1 and 3, respectively;
FFU, fluorescent focus units;
RBD, receptor-binding domain;
MEL, murine erythroleukemia;
mAb, monoclonal
antibody;
UTR, untranslated region;
SPR, surface plasmon resonance;
PBS, phosphate-buffered saline;
D-PBS, Dulbecco's phosphate-buffered
saline;
SLL, -sialyllactose;
pfu, plaque-forming unit(s);
MOI, multiplicity of infection.
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REFERENCES |
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