Previous Article | Next Article 
Journal of Virology, January 2000, p. 644-651, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Large-Plaque Mutants of Sindbis Virus Show Reduced
Binding to Heparan Sulfate, Heightened Viremia, and Slower Clearance
from the Circulation
Andrew P.
Byrnes1 and
Diane E.
Griffin1,2,*
Departments of Molecular Microbiology and
Immunology1 and of Medicine and
Neurology,2 Johns Hopkins University School of
Hygiene and Public Health, Baltimore, Maryland 21205
Received 7 June 1999/Accepted 7 October 1999
 |
ABSTRACT |
Laboratory strains of Sindbis virus must bind to the negatively
charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with
a reduced ability to bind to heparan sulfate. Sequencing of these
mutants revealed changes of positively charged amino acids in putative
heparin-binding domains of the E2 glycoprotein. Recombinant viruses
were constructed with these changes as single amino acid substitutions
in a strain Toto 1101 background. All exhibited decreased binding to
heparan sulfate and had larger plaques than Toto 1101. When injected
subcutaneously into neonatal mice, large-plaque viruses produced
higher-titer viremia and often caused higher mortality. Because
circulating heparin-binding proteins are known to be rapidly
sequestered by tissue heparan sulfate, we measured the kinetics of
viral clearance following intravenous injection. Much of the parental
small-plaque Toto 1101 strain of Sindbis virus was cleared from the
circulation by the liver within minutes, in contrast to recombinant
large-plaque viruses, which had longer circulating half-lives. These
findings indicate that a decreased ability to bind to heparan sulfate
allows more efficient viral production in vivo, which may in turn lead
to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to
heparan sulfate, these results may be generally applicable to the
pathogenesis of such viruses.
| |
INTRODUCTION |
The alphaviruses are RNA viruses
which are carried by hematophagous insects, such as mosquitoes, and can
infect a wide variety of mammalian and avian hosts. In vertebrates,
they can replicate extremely rapidly and cause high-titer viremia,
which allows transmission of the virus to new mosquitoes. Sindbis
virus (SV) is a particularly well-studied alphavirus which causes
a mild rash and arthritis in humans but can cause fatal
encephalomyelitis in mice.
The cell surface receptors which allow alphaviruses such as SV to
infect such a broad variety of species have not yet been conclusively
determined, but it has recently been shown that SV can attach to
heparan sulfate (HS), a negatively charged glycosaminoglycan expressed
on many types of cells (4, 23, 32). Sulfated glycosaminoglycans on the cell surface and in the extracellular matrix
normally bind a wide variety of growth factors, chemokines, enzymes,
and matrix components (30, 45) but are also important in the
attachment of a number of bacteria, protozoa, and viruses (42). Proteins typically bind electrostatically to HS by use of stretches of positively charged amino acids such as Lys and Arg, and
attachment of SV to HS is presumably mediated in the same fashion.
Although the use of cell surface HS greatly increases the efficiency of
SV attachment, it is not absolutely required for infection, and a
distantly related Alphavirus, Ross River virus,
does not bind HS at all (4). It has been proposed that the
ability of SV to bind HS could be an adaptation which arose in
laboratory strains during repeated passaging in tissue culture and that
wild-type strains of SV might not bind well to HS (32). Nevertheless, SV joins a growing number of viruses which have been
shown to bind HS, including many herpesviruses, human immunodeficiency virus type 1 (HIV-1), dengue virus, adeno-associated virus type 2, respiratory syncytial virus, foot-and-mouth disease virus (FMDV), human
papillomavirus type 11, and vaccinia virus (6, 16, 18, 26, 33, 38,
46, 54). A study of the relevance of HS to the pathogenesis of SV
may therefore lead to parallel insights into the in vivo behavior of
other HS-binding viruses.
The alphavirus virion structure is relatively simple, with an outer
lipid layer which is derived from the host cell membrane and which
contains 240 copies of the viral E1 and E2 glycoproteins (44). The E2 glycoprotein is synthesized as a larger
precursor which is cleaved into E2 and another short protein, E3, which remains associated with the virion for some alphaviruses, although not
for SV. Inside the virion is an icosahedral capsid which surrounds the
11.7-kb positive-sense RNA genome. During infection, the E2 glycoprotein is largely responsible for binding to cells. After the
virus is endocytosed, an acid-induced rearrangement of the glycoproteins exposes a hydrophobic domain of E1, allowing fusion of
the viral and cellular membranes and penetration of the capsid into the cytoplasm.
Pharmacokinetic studies have shown that when HS-binding proteins are
injected intravenously, they are rapidly cleared from the circulation
through binding to tissue HS (27, 28). There is reason to
suspect that HS-binding viruses may behave in the same manner. A number
of studies have shown that alphavirus strains have different clearance
rates after intravenous injection, with small-plaque (SP) variants
typically having shorter half-lives than large-plaque (LP) variants
(20, 22, 40), but the reason for these findings has been
unknown. We have proposed that binding to HS controls both the plaque
size and the circulating half-life and that SP variants are cleared
more quickly from the circulation because they bind better to HS
(4). This characteristic may contribute to lower levels of
viremia during infection with SP variants and consequent lower
mortality rates. The present study was undertaken to explore this
hypothesis, with the additional aim of defining the regions of the SV
glycoproteins which are involved in binding to HS.
| |
MATERIALS AND METHODS |
Viruses and cells.
BHK-21 cells were grown in Dulbecco's
modified Eagle medium containing 10% heat-inactivated fetal calf serum
and 50 µg of gentamicin per ml. CHO-K1 and
glycosaminoglycan-deficient pgsA-745 cells (10) were grown
in Ham's F-12 medium with the same supplements. SV strains Toto 1101 (41) and AR339 were grown and passaged on BHK cells. Plaque
assays were performed as previously described (4) with BHK
cells under 0.6% Bacto Agar (Difco, Detroit, Mich.) or agarose (Gibco
BRL, Gaithersburg, Md.).
Site-directed mutagenesis of Toto 1101 was performed with a 1.2-kb
StuI/BssHII fragment by use of the GeneEditor
mutagenesis system (Promega, Madison, Wis.). After being cloned into
pToto1101, the entire 1.2-kb region was verified by sequencing.
Plasmids were linearized with XhoI, and capped RNA was
transcribed with an Invitroscript CAP kit (Invitrogen, Carlsbad,
Calif.). RNA was electroporated into 5 × 106 pgsA-745
cells, and virus was allowed to grow for 2 days in Ham's F-12 medium
containing 10% heat-inactivated fetal calf serum. Titers of stocks
were determined by a plaque assay with BHK cells under agar. For
measuring plaque size, plates were overlaid with either agar or
agarose, and after 2 days, the diameters of at least 15 plaques were
measured to the nearest 0.5 mm.
[
35S]Met-Cys-labeled viral stocks were grown on BHK cells
as described previously (
4) following infection with
unpassaged
pgsA-745-derived stocks at a multiplicity of infection of
0.5
to 1. Unlabeled high-titer viral stocks for intravenous clearance
studies were grown in a similar manner. Both labeled and unlabeled
stocks were purified as described previously (
4) by
pelleting
with polyethylene glycol, banding on a potassium tartrate
gradient,
and pelleting through a 15% sucrose
cushion.
Binding of radiolabeled virus to CHO and pgsA-745 cell monolayers at
4°C was determined as previously described (
4) in
phosphate-buffered saline (PBS) (pH 7.2) with 0.5 mM MgCl
2,
0.5
mM CaCl
2, and 0.5% bovine serum
albumin.
Heparin-Sepharose chromatography.
Prepacked 1-ml
heparin-Sepharose HiTrap columns (Amersham Pharmacia Biotech,
Piscataway, N.J.) were equilibrated with 10 ml of 50 mM NaCl-5 mM
phosphate (pH 7.5)-0.5% bovine serum albumin. This step was followed
by the addition of 1 ml of the same buffer containing 100,000 cpm of
35S-labeled virus at a rate of 1 ml/min and collection of
1-ml fractions. After 10 ml of the same 50 mM NaCl buffer was added,
the virus was eluted with a 40-ml linear gradient from 50 to 500 mM
NaCl. Any remaining virus was removed with 10 ml of 0.5% sodium
dodecyl sulfate. Fractions were counted by liquid scintillation
spectroscopy, and the NaCl concentration of the peak fraction was
determined with a conductivity meter.
ELISA.
Half-area 96-well plates were coated with
polyethylene glycol-precipitated virus at 0.6 µg per well. Monoclonal
antibodies (MAb) reactive against the SV E2 glycoprotein were added to
duplicate wells as 1:1,000 dilutions of ascitic fluid. Two MAb directed against the E2c epitope, R6 (37) and G5 (51),
along with 202, an antibody reactive with the E2ab epitope
(36), and 3E1, a negative control MAb against herpes simplex
virus, were used. The enzyme-linked immunosorbent assay (ELISA) was
developed with a rabbit anti-mouse antibody conjugated to horseradish
peroxidase, and samples were reacted with o-phenylenediamine
for 30 min at room temperature. Optical densities at 450 nm of <0.1
were considered negative, and values of >0.3 were considered positive.
Mice.
Alpha/beta interferon (IFN-
/
) receptor-deficient
(A129) mice were obtained from B&K Universal (Hull, United Kingdom) and bred in a specific-pathogen-free facility at Johns Hopkins University. Antibody-deficient (µMT) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Viral virulence was determined with 2-day-old litters of CD-1 mice obtained from Charles River Laboratories (Wilmington, Mass.). Mice were injected subcutaneously with 1,000 PFU
of unpassaged pgsA-745-grown virus in 30 µl of Hank's balanced salt
solution and were monitored daily for 15 days. At least four litters of
mice (total of at least 38 mice) were used per virus.
For intravenous clearance studies, 5- to 7-week-old male ICR mice
(Taconic, Germantown, N.Y.) weighing 30 to 40 g were anesthetized
intraperitoneally with tribromoethanol (Avertin; 0.5 mg per g
of body
weight) and kept anesthetized until the end of the experiment.
Mice
were injected intravenously via the tail vein with about
10
7 PFU of gradient-purified virus in 100 µl of PBS.
Blood was collected
from the retro-orbital plexus at 2, 5, 10, 20, 30, and 45 min
postinjection by use of a 20-µl capillary tube. Blood was
allowed
to clot at room temperature, and the amount of virus in the
serum
was determined by a plaque assay with BHK cells. The clearance
curves were fitted to the equation
V =
Ae
t/
+
Be
t/
, where
V is the viral
concentration in PFU per milliliter and
t is the time
postinjection. Weighted (1/
V) nonlinear regression
was
performed with SigmaPlot (SPSS, Chicago, Ill.). The viral
concentrations were normalized by dividing them by
V0, the concentration
of virus at 0 min
postinjection, which was calculated according
to the above
equation.
Alternatively, ICR mice were injected intravenously with approximately
150,000 cpm of
35S-labeled purified virus in 100 µl of
PBS. At 30 min postinjection,
mice were exsanguinated by perfusion with
PBS. Various organs
were collected and dissolved in 5 M NaOH for 4 to
5 h at 70°C.
Following the addition of Liquiscint (National
Diagnostics, Atlanta,
Ga.) and neutralization with glacial acetic acid,
counts were
determined by liquid scintillation
spectroscopy.
| |
RESULTS |
Derivation of LP viruses.
Agar contains a sulfated
polysaccharide which interferes with the efficient attachment of
certain viruses to cells (49, 50). Agarose is a purified
form of agar which does not contain sulfated polysaccharides. We have
hypothesized that viruses which bind strongly to HS are impaired by the
sulfated polysaccharide in agar and therefore have a smaller plaque
size under agar than they do under agarose (4). In contrast,
the finding that the plaque size of a virus under agar is large and
essentially equal to the plaque size under agarose may be evidence that
a virus binds less well to glycosaminoglycans. We noticed that within a
few days after infection of mice with SP strains of SV, such as Toto
1101 and AR339, serum frequently had a mixture of SP and LP viruses
when assayed on BHK monolayers under agar. The LP mutants presumably
enjoy a selective advantage during infection (see below). For these
experiments, it was helpful to use models with long-lasting and
high-titer serum viremia, such as mice deficient in antibody or the
type I interferon receptor, although infection of normal neonatal mice
also resulted in LP mutants. Even so, LP viruses were not found in
every animal, and the percentage of LP viruses in the serum of any
given mouse was variable, ranging from a small percentage to 80%.
LP viruses from individual mice were isolated, plaque purified, and
expanded on BHK cells. RNA was isolated from infected
cells and
amplified by reverse transcription-PCR for complete
sequencing of the
E2, 6K, and E1 genes and partial sequencing
of the E3 gene, including
the furin protease cleavage site at
the E3-E2 junction. 6K is a short
hydrophobic protein that is
present in the virion lipid membrane in
small amounts and is not
expected to interact with cellular receptors.
Every LP isolate
had at least one amino acid change in the E2
ectodomain (Table
1), while no coding
changes in the E1, 6K, or E3 regions were
found. In some cases, a
second LP isolate from the same animal
was sequenced; these pairs of
isolates always had identical mutations.
Preliminary characterization of LP isolates indicated a reduced ability
to bind heparin (data not shown). To examine the effect
of specific E2
mutations in the absence of other potential changes
in unsequenced
regions of the viral genome, individual codon changes
were introduced
into plasmid pToto1101 in order to transcribe
infectious SV RNA
(
41). When more than one mutation was found
in an individual
virus, the change involving the loss of a positively
charged amino acid
was chosen (Table
2), as such residues might
participate directly in
binding to sulfates on HS. Mutation of
the Lys at residue 76 of E2 was
found in two separate isolates,
one with a change to Asn and one with a
change to Glu (Table
1).
Because a change of Lys 76 to Thr has
previously been reported
to occur during selection for budding in
cultured cells under
low-ionic-strength conditions (
34), we
also constructed a virus
containing Thr at this
position.
Transfection and passaging of non-HS-binding SV variants on BHK cells
can result in the rapid selection of HS-binding mutant
viruses, which
are at a selective advantage in tissue cultures
because they bind much
better to BHK cells (
32). To minimize
such selection
pressure, we transfected viral RNA into pgsA-745
cells (which do not
synthesize HS) and produced high-titer stocks
by infecting BHK cells
with the unpassaged virus at a relatively
high multiplicity of
infection (see Materials and
Methods).
All seven of the recombinant viruses produced plaques under agar which
were significantly larger than plaques made by the
parental strain Toto
1101 (Table
2), indicating that the LP
phenotypes
of the original viral isolates were due solely to mutations
in
the E2 glycoprotein.
Viral binding to heparin, HS, and MAb.
Radiolabeled, purified
preparations of Toto 1101 and the seven recombinant viruses were
examined by heparin-Sepharose chromatography. Heparin is a more highly
sulfated glycosaminoglycan than HS, but the carbohydrate structure is
otherwise similar. Mutated viruses were eluted from heparin-Sepharose
columns at a range of NaCl concentrations, but all were eluted earlier
than Toto 1101, indicating weaker binding to heparin (Fig.
1 and Table 2). All three viruses with
substitutions for Lys 76 bound poorly to heparin. However, the virus
containing the polar amino acid Asn at position 76 had the best binding
of the three
heparin-binding sites sometimes contain Asn or Gln
residues which interact with heparin through hydrogen bonds
(15). Virus with Thr 76 bound more poorly to heparin than
virus with Asn 76, and the negatively charged Glu 76 mutant bound most
poorly of all. This result may indicate that the residue at position 76 interacts directly with sulfates on heparin and that the overall charge
of the E2 glycoprotein at this position is important in determining the
strength of the interaction.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
Viral binding to heparin. Radiolabeled SV was applied to
heparin-Sepharose and eluted with a 50 to 500 mM NaCl gradient. Elution
at a higher NaCl concentration indicates stronger binding to heparin.
Eluted virus is intact and fully infectious. (Five other viruses are
not shown; see Table 2 for results.)
|
|
When viral binding to monolayers of CHO cells was examined, the
percentage of virus bound by the monolayers correlated well
(
r2, 0.76) with the ability of the virus to bind
to heparin (Fig.
2). CHO cells express HS
and chondroitin sulfate, but the CHO-derived
pgsA-745 cell line
expresses neither of these glycosaminoglycans
(
10). When
binding to pgsA-745 cells was examined, viruses such
as Toto 1101 and
R157H, which bound strongly to CHO cells, bound
much less well to
pgsA-745 cells. In contrast, viruses K76E, K76T,
and K76N bound equally
well to CHO and pgsA-745 cells, indicating
that they were essentially
unable to bind to cellular HS. Surprisingly,
however, there was a
correlation (
r2, 0.96) between heparin binding
and binding to pgsA-745 cells
(Fig.
2). In other words, even in the
complete absence of cellular
HS, viruses such as Toto 1101 and R157H
were two- to threefold
better at binding to pgsA-745 cells than K76E,
K76T, and K76N
were. This result may indicate that disrupting the
ability of
the virus to bind to heparin and HS also decreases the
ability
to bind to other, nonglycosaminoglycan receptors.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 2.
Viral binding to cultured cells. Radiolabeled SV was
allowed to bind to CHO or glycosaminoglycan-deficient pgsA-745 cells at
4°C for 2 h. The mean binding ± standard deviation for
three replicates is shown. The data along the x axis are
taken from Table 2.
|
|
Two of the mutations found in our LP viruses, N62D and K159E, have been
previously described by Pence et al. during selection
for antibody
escape mutants which fail to bind MAb R6, a neutralizing
antibody
against the E2c epitope (
39). Because we established
that
these two amino acid changes also decrease binding to heparin,
we
examined whether a lack of reactivity to MAb R6 was a general
feature
of viruses with impaired binding to heparin. Of the seven
mutants,
however, only N62D and K159E failed to bind to MAb R6
(Table
2),
indicating that this was not the case. All eight viruses
bound equal
amounts of MAb G5 (another antibody against the E2c
epitope) and MAb
202, an antibody against the E2ab
epitope.
Behavior in mice.
Heparin-binding proteins tend to have very
short half-lives in the circulation, being rapidly sequestered by
tissue HS, particularly in the liver, which has the most highly
sulfated HS of any organ (2, 35, 53, 55). It has been
reported in several studies that plaque variants of alphaviruses are
cleared at variable rates from the circulation after an intravenous
injection, with LP variants typically having longer half-lives
(19, 20, 40). Because it is now apparent that one of the
factors contributing to increased plaque size is a decreased ability to
bind to sulfated polysaccharides, we reexamined the elimination of
virus from the circulation. Purified viral preparations were injected
intravenously as a bolus, and blood was collected at intervals for 45 min. The parental virus, Toto 1101, was cleared from the circulation
much faster than any of the seven viruses having reduced heparin
affinity (Fig. 3). All viruses were
eliminated with biphasic kinetics (see Discussion). The clearance of
infectious virus shown here represents actual physical removal of the
virus from the circulation and is not due merely to inactivation of
infectivity. Previous studies have shown that alphaviruses, regardless
of plaque size, are not inactivated by serum or whole blood (22,
40), and clearance of radiolabeled alphaviruses from the
circulation has been shown to follow kinetics identical to the
clearance of PFU (20, 21).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Kinetics of viral clearance. Mice were injected
intravenously with a 100-µl bolus of purified virus. Serum was
collected, and titers were determined by a plaque assay with BHK cells
at various times postinjection; V/V0 indicates
the fraction of virus remaining. Each point represents the mean ± standard deviation for three mice. Curves were fitted by nonlinear
regression as described in Materials and Methods. Symbols:  , K76T;
 , K230M;  , N62D;  , K159E;  , K76E;  , K76N;  , R157H;
 , Toto 1101.
|
|
In order to determine the organ distribution of virus following
clearance from the circulation, various radiolabeled viruses
were
injected intravenously, and selected organs were collected
after
perfusion of the mice 30 min later. For Toto 1101, about
half of the
injected counts were found in the liver, with a much
smaller amount
being associated with the spleen (Table
3). When
K159E and K76E were injected,
the liver contained significantly
smaller amounts than had been found
with Toto 1101 (
P, <0.05,
as determined by an analysis of
variance followed by a Tukey test).
K159E and K76E were also found in
the spleen, although the amounts
were highly variable, with no
significant differences compared
to the amount of Toto 1101. For all
three viruses, little radioactivity
could be found in the kidneys,
lungs, or brain. Not all of the
injected counts were recovered
for
K159E and K76E, 7 to 9% of
the virus was still in the circulation at
this time (Fig.
3),
and the remaining virus was presumed to be
distributed widely
throughout the body. Another
Alphavirus,
Venezuelan equine encephalitis virus (VEE virus), shows a
similar organ distribution after intravenous
injection (
20).
Toto 1101 and each of the seven recombinant viruses were injected
subcutaneously into 2-day-old mice, and serum titers were
assayed over
the following 2 days (Fig.
4A). In every
case, mice
infected with an LP mutant had significantly higher titers
than
mice infected with Toto 1101. However, when the peak serum titer
was plotted against the strength of association with heparin,
no
obvious relationship between these two factors was apparent
(Fig.
4B).
Thus, while reducing the ability of the viruses to
bind to HS always
led to higher viremia, the mutations may have
had other effects on the
ability of the viruses to replicate.
It should be pointed out that
plaque assays are an imperfect measure
of the amount of a virus when
the viruses being compared do not
bind equally well to HS. Viruses such
as Toto 1101 and R157H bind
much better to BHK cells and will have a
lower particle/PFU ratio
than viruses such as K76E.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4.
Serum titers. (A) CD-1 pups (2 days old) were infected
subcutaneously with 1,000 PFU of virus, and serum titers were assessed
at various times by a plaque assay (mean for three mice at each time
point; error bars are not shown). All LP viruses retained their LP
phenotype. A two-way analysis of variance showed a significant
(P, <0.0001) effect of viral strain on titer, and titers of
Toto 1101 were lower than those of all seven mutant viruses when
compared by Scheffe's test (P, <0.05). (B) Plotting of
peak titer (mean ± standard deviation) versus elution from
heparin-Sepharose failed to indicate a simple correlation between virus
replication in vivo and the strength of binding to heparin.
|
|
Four of the seven mutants (N62D, K76E, R157H, and K230M) produced
significantly greater mortality than Toto 1101 when injected
subcutaneously into 2-day-old mice (Fig.
5). The three remaining
mutants (K76T,
K76N, and K159E) had survival curves that were
approximately similar to
that of Toto 1101. Thus, even though
all seven mutants produced
significantly higher viremia than Toto
1101, only four caused
significantly increased mortality.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 5.
Survival curves. Litters of 2-day-old CD-1 pups were
infected subcutaneously with 1,000 PFU of virus, and survival was
monitored for 15 days. R157H, K230M, N62D, and K76E produced
significantly greater mortality than Toto 1101. Survival curves were
compared pairwise against Toto 1101 by use of the log rank test with a
threshold for significance (P) of <0.0071; the Bonferonni
correction for multiple comparisons was used.
|
|
| |
DISCUSSION |
In this study, we derived a panel of viruses with different
abilities to bind HS, both to define the HS-interacting regions of the
SV glycoproteins and to find out how the ability to bind HS influences
infection in vivo. Because the plaque size of SV is affected by
interaction with sulfated polysaccharides in agar, we were able to
easily screen for viruses with a reduced ability to bind HS.
Low-HS-binding LP mutants arose spontaneously during infection in mice
and had alterations scattered through a wide region of the E2
glycoprotein. Viruses with these changes in E2 were cleared more slowly
from the blood, caused higher viremia, and often induced greater mortality.
Binding of E2 to HS.
In the absence of detailed structural
information about the alphavirus E2 glycoprotein, it is difficult to
demonstrate that any particular amino acid is part of a binding site
for HS or even that the amino acid is exposed on the surface of the
virion. The mutations described here could, for example, alter the
tertiary structure of E2 and obscure an HS-binding site located
elsewhere on E2 or E1. Nevertheless, four of the five mutated positions (K76, R157, K159, and K230) encode Lys or Arg. Because interactions between HS and proteins are primarily electrostatic and involve negatively charged sulfates binding to positively charged amino acids,
these four residues have the potential to interact directly with HS.
The remaining mutation in this study, N62D, did not involve the loss of
a positively charged amino acid, but the gain of a negatively charged
Asp might interfere with binding to HS by electrostatic repulsion.
It is likely that the Lys at position 76 directly participates in
binding to HS, since replacing Lys 76 with negatively charged
Glu
resulted in poorer binding to heparin than was seen for viruses
with
either Asn or Thr at this position (Table
2). This result
suggests that
the charge of the amino acid at position 76 may
be an important
determinant of the strength of the interaction
between E2 and heparin.
An analogous mutation in VEE virus, changing
the wild-type Glu 76 to
Lys, results in an SP virus which replicates
to lower titers in vivo
and causes less mortality (
7,
13).
Klimstra et al. have shown that mutations at additional E2 residues can
alter the binding of SV to HS (
32). Many strains
of SV have
a Lys at position 70 of E2, and changing this residue
to Glu leads to a
decreased ability to bind to HS. Also, substitution
of an Arg for the
Ser at position 114 results in an increase in
binding to HS
(
32). The involvement of positively charged residues
again
suggests that these may contribute directly to binding of
HS rather
than indirectly by altering the tertiary structure of
E2. In addition,
although E3 is normally shed from the virion,
the positively charged
furin protease cleavage site at the C terminus
of E3 can mediate
binding to HS in cleavage-defective mutants
when E3 remains attached to
E2 (
31).
It may appear odd that mutations over such a wide stretch of E2,
spanning residues 62 to 230, can affect binding to HS, especially
given
that many HS-binding proteins have very short HS-binding
sites
containing clusters of Lys and Arg residues (
15). However,
HS-binding sites can also be formed when positively charged residues
are brought into proximity in the tertiary or quaternary structure
of a
protein. Indeed, it has recently been shown that the HS-binding
site on
the surface of FMDV virions lies at the intersection of
three different
capsid proteins (
11). Furthermore, the finding
of a widely
distributed HS-binding site on E2 is compatible with
the fact that the
E2c epitope is conformational and covers a large
stretch of E2. Binding
of MAb R6 to this epitope is affected by
mutations in residues 62, 96, 114, and 159, which are distant
from each other in the primary protein
sequence but are likely
to be located near each other in the
three-dimensional folded
structure of E2 (
37,
39).
Viral behavior in vivo.
Although numerous viruses bind to HS
and it is easy to show that this activity increases the efficiency of
viral attachment to cultured cells, almost no studies have examined how
binding to HS influences infection in vivo. Sa-Carvalho et al. have
shown that variants of FMDV which bind well to HS are attenuated in cattle, showing a decreased ability to spread from the site of inoculation (43). We (4) and others
(32) have proposed that the ability of SV to bind HS may
cause attenuation in vivo in a similar manner.
Fortunately, a great deal is known about how HS-binding proteins behave
in vivo. Pharmacokinetic studies on HS-binding proteins,
such as
bactericidal/permeability-increasing protein, extracellular-superoxide
dismutase, and hepatocyte growth factor/scatter factor, have
demonstrated
rapid biphasic clearance from the circulation after
intravenous
injection (
1,
14,
28). The biphasic decay can be
modeled
as the sum of two exponential equations (
12). The
early, rapid
phase of clearance is strongly influenced by binding to
HS; clearance
during this phase can be decreased by coinjecting heparin
(
27,
52), digesting tissue HS with intravenous heparinase
(
25),
or mutating basic residues so that the protein is no
longer able
to bind HS (
14,
28). Because the liver contains
large amounts
of highly sulfated HS (
35), a large percentage
of the protein
removed from the circulation can be found in this organ
(
2,
25,
27,
53,
55).
A number of investigations into the clearance of alphaviruses from the
circulation were performed 20 to 30 years ago; these
included studies
on SV (
40), western equine encephalitis virus
(
19), and VEE virus (
20-22). All showed that SP
variants were
typically cleared faster after an intravenous injection
than LP
variants. One of these studies demonstrated the accumulation of
VEE virus in the liver, with virions deposited in sinusoids and
the
spaces of Disse, as well as within vacuoles of Kupffer cells
(
20). Given what is now known about the clearance of
HS-binding
proteins from the circulation and given that plaque size can
be
a marker for the ability to bind HS, it seems likely that the
differences in clearance rates in these studies were due to differences
in viral binding to
HS.
The seven mutant viruses in the present study all produced larger
plaques under agar than the parental virus Toto 1101 and
showed less
binding to heparin and cellular HS. All of these mutants
also were
cleared more slowly from the circulation and caused
higher viremia than
Toto 1101. These findings together with what
is known about the
behavior of HS-binding proteins in vivo provide
strong evidence that
the ability to bind HS has a negative impact
on SV production in vivo.
It is interesting to note that, even
though the viruses showed a broad
range of ability to bind heparin
and cellular HS, the plaque size and
clearance from the circulation
both showed threshold behavior
Toto
1101 had SPs and was cleared
quickly from the circulation, but all of
the LPs were approximately
the same size, and all of the LP viruses
were cleared at about
the same relatively slow
rate.
In addition to accelerated clearance, another mechanism which might
prevent HS-binding viruses from achieving high viremia
is interaction
with HS in the extracellular matrix near the site
of viral production.
When extracellular-superoxide dismutase is
injected subcutaneously or
intramuscularly, it diffuses away from
the injection site far more
slowly than truncated variants of
the enzyme which do not bind HS
(
29). The equilibrium amount
of virus in the blood is a
function of both the rate of release
of new virus into the circulation
and the rate of clearance, and
both of these may be decreased if the
virus can bind
HS.
In spite of the fact that all seven mutant viruses produced
significantly higher viremia than Toto 1101 after subcutaneous
injection in suckling mice, only four caused significantly greater
mortality, suggesting that high viremia may be only one of the
factors
necessary for high virulence. It should be noted that
the K159E
mutation, which did not increase virulence in our study,
did cause
increased virulence in another viral clone (
39), so
the
background strain of the virus modulates the effects of these
mutations
as
well.
Laboratory strains of SV have typically undergone multiple passages in
tissue cultures. Klimstra et al. demonstrated that
passaging of a
low-HS-binding strain can quickly select for mutants
with increased
affinity for HS and proposed that wild-type strains
of SV may not bind
well to HS (
32). Viruses which bind strongly
to HS not only
may have decreased virulence but also may have
trouble achieving high
enough viremia to allow transmission to
mosquitoes. However, the R157H
and N62D mutants in our study retained
some ability to bind to HS on
cultured cells and yet still caused
high viremia and high mortality.
Further study of unpassaged wild-type
isolates will be required to
determine how well natural strains
of SV bind
HS.
Relevance to other viruses.
The general conclusion that strong
binding to a ubiquitous carbohydrate such as HS causes attenuation in
vivo may apply only to viruses which cause plasma viremia and to
instances in which viral spread through the circulation contributes to
dissemination within the infected host. High viremia is also an
important factor in transmission from host to host for insect-borne
arboviruses, such as SV and dengue. In contrast, for viruses such as
herpes simplex virus type 1, infection is spread primarily from cell to
cell and strong binding to HS is not necessarily deleterious.
FMDV is a picornavirus that infects cattle. It causes viremia, and
there is evidence that spread through the bloodstream is
important in
the dissemination of the virus within the animal
(
48).
Wild-type isolates of FMDV do not bind to HS, but upon
tissue culture
passaging, SP variants with the ability to bind
to HS arise (
18,
43). HS-binding variants attach better to
cultured cells but are
attenuated in mice and cattle (
24,
43),
apparently because
of a reduced ability to spread from the site
of inoculation. After
injection of cattle with very high doses
of an attenuated HS-binding
variant, disease and systemic dissemination
of the virus can be seen
but are due to the development of non-HS-binding
LP revertants
(
43). Thus, the virus is under strong selective
pressure to
not bind HS during infection in vivo. A study of the
clearance of
virulent FMDV from the circulation has been done
(
47); we
would predict that avirulent, HS-binding variants would
be cleared
considerably
faster.
HIV-1 uses CD4 as a receptor and various chemokine receptors as
coreceptors. During the early stages of infection, viruses
typically
use CCR5 as a coreceptor and infect monocytes. At the
point of
progression to AIDS, there is typically a switch, and
T-cell-tropic
viruses which use CXCR4 or both CXCR4 and CCR5 arise.
Interestingly,
only variants that use CXCR4 or both CXCR4 and
CCR5 can bind well to
HS, and this activity correlates with an
increase in the positive
charge in the V3 loop (
3,
52). Studies
on simian
immunodeficiency virus and HIV-1 have found very rapid
clearance of
virus from the circulation following intravenous
injection of virus
into monkeys (
17,
56). In order to obtain
a complete
understanding of the dynamics of viral behavior, it
will be necessary
to study whether clearance from the circulation
is changed by the
ability of some strains to bind
HS.
Finally, viruses that bind to other ubiquitously present carbohydrates
can also show attenuation in vivo if they bind too
avidly. Murine
polyomavirus binds to sialic acid but does not
encode a neuraminidase
like influenza virus. Polyomavirus SP variants
bind better to cultured
cells than LP variants because they attach
more avidly to branched
sialic-acid-containing oligosaccharides
(
5); the difference
in plaque size is due to binding of sialylated
oligosaccharides in the
overlay, analogous to the effect of agar
sulfated polysaccharides on
SV. When virus is injected intraperitoneally,
SP viruses cause more
tumors in the peritoneum than in the kidney,
while the distribution of
tumors is reversed for LP viruses (
8).
This pattern is due
to the fact that LP variants can cause widely
disseminated infection,
whereas SP variants cannot (
9). Thus,
there are multiple
ways by which receptor binding influences the
pathogenesis of viral
infections in
vivo.
| |
ACKNOWLEDGMENTS |
This work was supported by a postdoctoral fellowship from the
National Multiple Sclerosis Society (to A.P.B.) and grant R01-NS18596 from the National Institutes of Health (to D.E.G.).
We thank Gwendolyn Binder and Karl Zheng for several of the viruses in
this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3459. Fax: (410) 955-0105. E-mail:
dgriffin{at}welchlink.welch.jhu.edu.
| |
REFERENCES |
| 1.
|
Abrahamson, S. L.,
H.-M. Wu,
R. E. Williams,
K. Der,
N. Ottah,
R. Little,
H. Gazzano-Santoro,
G. Theofan,
R. Bauer,
S. Leigh,
A. Orme,
A. H. Horwitz,
S. F. Carroll, and R. L. Dedrick.
1997.
Biochemical characterization of recombinant fusions of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein.
J. Biol. Chem.
272:2149-2155[Abstract/Free Full Text].
|
| 2.
|
Bauer, R. J.,
K. Der,
N. Ottah-Ihejeto,
J. Barrientos, and A. H. C. Kung.
1997.
The role of liver and kidney on the pharmacokinetics of a recombinant amino terminal fragment of bactericidal/permeability-increasing protein in rats.
Pharm. Res.
14:224-229[CrossRef][Medline].
|
| 3.
|
Bhattacharyya, D.,
B. R. Brooks, and L. Callahan.
1996.
Positioning of positively charged residues in the V3 loop correlates with HIV type 1 syncytium-inducing phenotype.
AIDS Res. Hum. Retrovir.
12:83-90[Medline].
|
| 4.
|
Byrnes, A. P., and D. E. Griffin.
1998.
Binding of Sindbis virus to cell surface heparan sulfate.
J. Virol.
72:7349-7356[Abstract/Free Full Text].
|
| 5.
|
Cahan, L. D.,
R. Singh, and J. C. Paulson.
1983.
Sialyloligosaccharide receptors of binding variants of polyoma virus.
Virology
130:281-289[CrossRef][Medline].
|
| 6.
|
Chen, Y.,
T. Maguire,
R. E. Hileman,
J. R. Fromm,
J. D. Esko,
R. J. Linhardt, and R. M. Marks.
1997.
Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.
Nat. Med.
3:866-871[CrossRef][Medline].
|
| 7.
|
Davis, N. L.,
N. Powell,
G. F. Greenwald,
L. V. Willis,
B. J. B. Johnson,
J. F. Smith, and R. E. Johnston.
1991.
Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone.
Virology
183:20-31[CrossRef][Medline].
|
| 8.
|
Diamond, L.
1964.
Cell transformation in vitro and tumor induction in vivo by large- and small-plaque polyoma virus.
Virology
23:73-80[CrossRef][Medline].
|
| 9.
|
Dubensky, T. W.,
R. Freund,
C. J. Dawe, and T. L. Benjamin.
1991.
Polyomavirus replication in mice: influences of VP1 type and route of inoculation.
J. Virol.
65:342-349[Abstract/Free Full Text].
|
| 10.
|
Esko, J. D.,
T. E. Stewart, and W. H. Taylor.
1985.
Animal cell mutants defective in glycosaminoglycan biosynthesis.
Proc. Natl. Acad. Sci. USA
82:3197-3201[Abstract/Free Full Text].
|
| 11.
|
Fry, E. E.,
S. M. Lea,
T. Jackson,
J. W. I. Newman,
F. M. Ellard,
W. E. Blakemore,
R. Abu-Ghazaleh,
A. Samuel,
A. M. Q. King, and D. I. Stuart.
1999.
The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.
EMBO J.
18:543-554[CrossRef][Medline].
|
| 12.
|
Gibaldi, M., and D. Perrier.
1975.
Pharmacokinetics, p. 45-96.
Marcel Dekker, Inc., New York, N.Y.
|
| 13.
|
Grieder, F. B.,
N. L. Davis,
J. F. Aronson,
P. C. Charles,
D. C. Sellon,
K. Suzuki, and R. E. Johnston.
1995.
Specific restrictions in the progression of Venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins.
Virology
206:994-1006[CrossRef][Medline].
|
| 14.
|
Hartmann, G.,
T. Prospero,
V. Brinkmann,
Ö. Ozcelik,
G. Winter,
J. Hepple,
S. Batley,
F. Bladt,
M. Sachs,
C. Birchmeier,
W. Birchmeier, and E. Gherardi.
1998.
Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.
Curr. Biol.
8:125-134[CrossRef][Medline].
|
| 15.
|
Hileman, R. E.,
J. R. Fromm,
J. M. Weiler, and R. J. Linhardt.
1998.
Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins.
Bioessays
20:156-167[CrossRef][Medline].
|
| 16.
|
Hsiao, J.-C.,
C.-S. Chung, and W. Chang.
1998.
Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain.
J. Virol.
72:8374-8379[Abstract/Free Full Text].
|
| 17.
|
Igarashi, T.,
C. Brown,
A. Azadegan,
N. Haigwood,
D. Dimitrov,
M. A. Martin, and R. Shibata.
1999.
Human immunodeficiency virus type 1 neutralizing antibodies accelerate clearance of cell-free virions from blood plasma.
Nat. Med.
5:211-216[CrossRef][Medline].
|
| 18.
|
Jackson, T.,
F. M. Ellard,
R. Abu Ghazaleh,
S. M. Brookes,
W. E. Blakemore,
A. H. Corteyn,
D. I. Stuart,
J. W. I. Newman, and A. M. Q. King.
1996.
Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.
J. Virol.
70:5282-5287[Abstract/Free Full Text].
|
| 19.
|
Jahrling, P. B.
1976.
Virulence heterogeneity of a predominantly avirulent western equine encephalitis virus population.
J. Gen. Virol.
32:121-128[Abstract/Free Full Text].
|
| 20.
|
Jahrling, P. B., and L. Gorelkin.
1975.
Selective clearance of a benign clone of Venezuelan equine encephalitis virus from hamster plasma by hepatic reticuloendothelial cells.
J. Infect. Dis.
132:667-676[Medline].
|
| 21.
|
Jahrling, P. B.,
D. E. Hilmas, and C. D. Heard.
1977.
Vascular clearance of Venezuelan equine encephalomyelitis viruses as a correlate to virulence for rhesus monkeys.
Arch. Virol.
55:161-164[CrossRef][Medline].
|
| 22.
|
Jahrling, P. B., and W. F. Scherer.
1973.
Growth curves and clearance rates of virulent and benign Venezuelan encephalitis viruses in hamsters.
Infect. Immun.
8:456-462[Abstract/Free Full Text].
|
| 23.
|
Jan, J.-T.,
A. P. Byrnes, and D. E. Griffin.
1999.
Characterization of a Chinese hamster ovary cell line developed by retroviral insertional mutagenesis that is resistant to Sindbis virus infection.
J. Virol.
73:4919-4924[Abstract/Free Full Text].
|
| 24.
|
Jensen, M. J., and D. M. Moore.
1993.
Phenotypic and functional characterization of mouse attenuated and virulent variants of foot-and-mouth disease virus type O1 Campos.
Virology
193:604-613[CrossRef][Medline].
|
| 25.
|
Ji, Z.-S.,
D. A. Sanan, and R. W. Mahley.
1995.
Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans.
J. Lipid Res.
36:583-592[Abstract].
|
| 26.
|
Joyce, J. G.,
J. S. Tung,
C. T. Przysiecki,
J. C. Cook,
E. D. Lehman,
J. A. Sands,
K. U. Jansen, and P. M. Keller.
1999.
The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.
J. Biol. Chem.
274:5810-5822[Abstract/Free Full Text].
|
| 27.
|
Karlsson, K., and S. L. Marklund.
1988.
Plasma clearance of human extracellular-superoxide dismutase C in rabbits.
J. Clin. Investig.
82:762-766.
|
| 28.
|
Karlsson, K.,
J. Sandström,
A. Edlund,
T. Edlund, and S. L. Marklund.
1993.
Pharmacokinetics of extracellular-superoxide dismutase in the vascular system.
Free Rad. Biol. Med.
14:185-190[CrossRef][Medline].
|
| 29.
|
Karlsson, K.,
J. Sandström,
A. Edlund, and S. L. Marklund.
1994.
Turnover of extracellular-superoxide dismutase in tissues.
Lab. Investig.
70:705-710[Medline].
|
| 30.
|
Kjellén, L., and U. Lindahl.
1991.
Proteoglycans: structures and interactions.
Annu. Rev. Biochem.
60:443-475[CrossRef][Medline].
|
| 31.
|
Klimstra, W. B.,
H. W. Heidner, and R. E. Johnston.
1999.
The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate.
J. Virol.
73:6299-6306[Abstract/Free Full Text].
|
| 32.
|
Klimstra, W. B.,
K. D. Ryman, and R. E. Johnston.
1998.
Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor.
J. Virol.
72:7357-7366[Abstract/Free Full Text].
|
| 33.
|
Krusat, T., and H.-J. Streckert.
1997.
Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells.
Arch. Virol.
142:1247-1254[CrossRef][Medline].
|
| 34.
|
Li, M.-L., and V. Stollar.
1995.
A mutant of Sindbis virus which is released efficiently from cells maintained in low ionic strength medium.
Virology
210:237-243[CrossRef][Medline].
|
| 35.
|
Lyon, M.,
J. A. Deakin, and J. T. Gallagher.
1994.
Liver heparan sulfate structure: a novel molecular design.
J. Biol. Chem.
269:11208-11215[Abstract/Free Full Text].
|
| 36.
|
Mendoza, Q. P.,
J. Stanley, and D. E. Griffin.
1988.
Monoclonal antibodies to the E1 and E2 glycoproteins of Sindbis virus: definition of epitopes and efficiency of protection from fatal encephalitis.
J. Gen. Virol.
70:3015-3022[Abstract/Free Full Text].
|
| 37.
|
Olmsted, R. A.,
W. J. Meyer, and R. E. Johnston.
1986.
Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals.
Virology
148:245-254[CrossRef][Medline].
|
| 38.
|
Patel, M.,
M. Yanagishita,
G. Roderiquez,
D. C. Bou-Habib,
T. Oravecz,
B. C. Hascall, and M. A. Norcross.
1993.
Cell-surface heparan sulfate proteoglycan mediates HIV-1 infection of T-cell lines.
AIDS Res. Hum. Retrovir.
9:167-174[Medline].
|
| 39.
|
Pence, D. F.,
N. L. Davis, and R. E. Johnston.
1990.
Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2.
Virology
175:41-49[CrossRef][Medline].
|
| 40.
|
Postic, B.,
C. J. Schleupner,
J. A. Armstrong, and M. Ho.
1969.
Two variants of Sindbis virus which differ in interferon induction and serum clearance. I. The phenomenon.
J. Infect. Dis.
120:339-347[Medline].
|
| 41.
|
Rice, C. M.,
R. Levis,
J. H. Strauss, and H. V. Huang.
1987.
Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.
J. Virol.
61:3809-3819[Abstract/Free Full Text].
|
| 42.
|
Rostand, K. S., and J. D. Esko.
1997.
Microbial adherence to and invasion through proteoglycans.
Infect. Immun.
65:1-8[Medline].
|
| 43.
|
Sa-Carvalho, D.,
E. Rieder,
B. Baxt,
R. Rodarte,
A. Tanuri, and P. W. Mason.
1997.
Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle.
J. Virol.
71:5115-5123[Abstract].
|
| 44.
|
Strauss, J. H., and E. G. Strauss.
1994.
The alphaviruses: gene expression, replication, and evolution.
Microbiol. Rev.
58:491-562[Abstract/Free Full Text].
|
| 45.
|
Stringer, S. E., and J. T. Gallagher.
1997.
Heparan sulphate.
Int. J. Biochem. Cell Biol.
29:709-714[CrossRef][Medline].
|
| 46.
|
Summerford, C., and R. J. Samulski.
1998.
Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.
J. Virol.
72:1438-1445[Abstract/Free Full Text].
|
| 47.
|
Sutmoller, P., and J. W. McVicar.
1976.
Pathogenesis of foot-and-mouth disease: clearance of the virus from the circulation of cattle and goats during experimental viraemia.
J. Hyg.
77:245-253.
|
| 48.
|
Sutmoller, P., and J. W. McVicar.
1976.
Pathogenesis of foot-and-mouth disease: the lung as an additional portal of entry of the virus.
J. Hyg.
77:235-243.
|
| 49.
|
Takemoto, K. K.
1966.
Plaque mutants of animal viruses.
Prog. Med. Virol.
8:314-348[Medline].
|
| 50.
|
Takemoto, K. K., and H. Liebhaber.
1961.
Virus-polysaccharide interactions. I. An agar polysaccharide determining plaque morphology of EMC virus.
Virology
14:456-462[CrossRef][Medline].
|
| 51.
|
Ubol, S.,
B. Levine,
S.-H. Lee,
N. S. Greenspan, and D. E. Griffin.
1995.
Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons.
J. Virol.
69:1990-1993[Abstract].
|
| 52.
|
Ugolini, S.,
I. Mondor, and Q. J. Sattentau.
1999.
HIV-1 attachment: another look.
Trends Microbiol.
7:144-149[CrossRef][Medline].
|
| 53.
|
Wells, M. J., and M. A. Blajchman.
1998.
In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans.
J. Biol. Chem.
273:23440-23447[Abstract/Free Full Text].
|
| 54.
|
WuDunn, D., and P. G. Spear.
1989.
Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.
J. Virol.
63:52-58[Abstract/Free Full Text].
|
| 55.
|
Yuge, T.,
A. Furukawa,
K. Nakamura,
Y. Nagashima,
K. Shinozaki,
T. Nakamura, and R. Kimura.
1997.
Metabolism of the intravenously administered recombinant human basic fibroblast growth factor, trafermin, in liver and kidney: degradation implicated in its selective localization to the fenestrated type microvasculatures.
Biol. Pharm. Bull.
20:786-793[Medline].
|
| 56.
|
Zhang, L.,
P. J. Dailey,
T. He,
A. Gettie,
S. Bonhoeffer,
A. S. Perelson, and D. D. Ho.
1999.
Rapid clearance of simian immunodeficiency virus particles from plasma of rhesus macaques.
J. Virol.
73:855-860[Abstract/Free Full Text].
|
Journal of Virology, January 2000, p. 644-651, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Knight, R. L., Schultz, K. L. W., Kent, R. J., Venkatesan, M., Griffin, D. E.
(2009). Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems. J. Virol.
83: 5640-5647
[Abstract]
[Full Text]
-
Adams, A. P., Aronson, J. F., Tardif, S. D., Patterson, J. L., Brasky, K. M., Geiger, R., de la Garza, M., Carrion, R. Jr., Weaver, S. C.
(2008). Common Marmosets (Callithrix jacchus) as a Nonhuman Primate Model To Assess the Virulence of Eastern Equine Encephalitis Virus Strains. J. Virol.
82: 9035-9042
[Abstract]
[Full Text]
-
Prestwood, T. R., Prigozhin, D. M., Sharar, K. L., Zellweger, R. M., Shresta, S.
(2008). A Mouse-Passaged Dengue Virus Strain with Reduced Affinity for Heparan Sulfate Causes Severe Disease in Mice by Establishing Increased Systemic Viral Loads. J. Virol.
82: 8411-8421
[Abstract]
[Full Text]
-
Lee, E., Lobigs, M.
(2008). E Protein Domain III Determinants of Yellow Fever Virus 17D Vaccine Strain Enhance Binding to Glycosaminoglycans, Impede Virus Spread, and Attenuate Virulence. J. Virol.
82: 6024-6033
[Abstract]
[Full Text]
-
Meisner, J., Szretter, K. J., Bradley, K. C., Langley, W. A., Li, Z.-N., Lee, B.-J., Thoennes, S., Martin, J., Skehel, J. J., Russell, R. J., Katz, J. M., Steinhauer, D. A.
(2008). Infectivity Studies of Influenza Virus Hemagglutinin Receptor Binding Site Mutants in Mice. J. Virol.
82: 5079-5083
[Abstract]
[Full Text]
-
Aguilar, P. V., Adams, A. P., Wang, E., Kang, W., Carrara, A.-S., Anishchenko, M., Frolov, I., Weaver, S. C.
(2008). Structural and Nonstructural Protein Genome Regions of Eastern Equine Encephalitis Virus Are Determinants of Interferon Sensitivity and Murine Virulence. J. Virol.
82: 4920-4930
[Abstract]
[Full Text]
-
Pierro, D. J., Powers, E. L., Olson, K. E.
(2007). Genetic determinants of Sindbis virus strain TR339 affecting midgut infection in the mosquito Aedes aegypti. J. Gen. Virol.
88: 1545-1554
[Abstract]
[Full Text]
-
Ryman, K. D., Gardner, C. L., Burke, C. W., Meier, K. C., Thompson, J. M., Klimstra, W. B.
(2007). Heparan Sulfate Binding Can Contribute to the Neurovirulence of Neuroadapted and Nonneuroadapted Sindbis Viruses. J. Virol.
81: 3563-3573
[Abstract]
[Full Text]
-
Lee, E., Wright, P. J., Davidson, A., Lobigs, M.
(2006). Virulence attenuation of Dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination.. J. Gen. Virol.
87: 2791-2801
[Abstract]
[Full Text]
-
Rubio, M.-P., Lopez-Bueno, A., Almendral, J. M.
(2005). Virulent Variants Emerging in Mice Infected with the Apathogenic Prototype Strain of the Parvovirus Minute Virus of Mice Exhibit a Capsid with Low Avidity for a Primary Receptor. J. Virol.
79: 11280-11290
[Abstract]
[Full Text]
-
Greene, I. P., Paessler, S., Austgen, L., Anishchenko, M., Brault, A. C., Bowen, R. A., Weaver, S. C.
(2005). Envelope Glycoprotein Mutations Mediate Equine Amplification and Virulence of Epizootic Venezuelan Equine Encephalitis Virus. J. Virol.
79: 9128-9133
[Abstract]
[Full Text]
-
Fry, E. E., Newman, J. W. I., Curry, S., Najjam, S., Jackson, T., Blakemore, W., Lea, S. M., Miller, L., Burman, A., King, A. M. Q., Stuart, D. I.
(2005). Structure of Foot-and-mouth disease virus serotype A1061 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J. Gen. Virol.
86: 1909-1920
[Abstract]
[Full Text]
-
Unno, Y., Shino, Y., Kondo, F., Igarashi, N., Wang, G., Shimura, R., Yamaguchi, T., Asano, T., Saisho, H., Sekiya, S., Shirasawa, H.
(2005). Oncolytic Viral Therapy for Cervical and Ovarian Cancer Cells by Sindbis Virus AR339 Strain. Clin. Cancer Res.
11: 4553-4560
[Abstract]
[Full Text]
-
Reddi, H. V., Kumar, A. S. M., Kung, A. Y., Kallio, P. D., Schlitt, B. P., Lipton, H. L.
(2004). Heparan Sulfate-Independent Infection Attenuates High-Neurovirulence GDVII Virus-Induced Encephalitis. J. Virol.
78: 8909-8916
[Abstract]
[Full Text]
-
Perri, S., Greer, C. E., Thudium, K., Doe, B., Legg, H., Liu, H., Romero, R. E., Tang, Z., Bin, Q., Dubensky, T. W. Jr., Vajdy, M., Otten, G. R., Polo, J. M.
(2003). An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector. J. Virol.
77: 10394-10403
[Abstract]
[Full Text]
-
Zhao, Q., Pacheco, J. M., Mason, P. W.
(2003). Evaluation of Genetically Engineered Derivatives of a Chinese Strain of Foot-and-Mouth Disease Virus Reveals a Novel Cell-Binding Site Which Functions in Cell Culture and in Animals. J. Virol.
77: 3269-3280
[Abstract]
[Full Text]
-
Wang, E., Brault, A. C., Powers, A. M., Kang, W., Weaver, S. C.
(2002). Glycosaminoglycan Binding Properties of Natural Venezuelan Equine Encephalitis Virus Isolates. J. Virol.
77: 1204-1210
[Abstract]
[Full Text]
-
Tseng, J.-C., Levin, B., Hirano, T., Yee, H., Pampeno, C., Meruelo, D.
(2002). In Vivo Antitumor Activity of Sindbis Viral Vectors. JNCI J Natl Cancer Inst
94: 1790-1802
[Abstract]
[Full Text]
-
Smit, J. M., Waarts, B.-L., Kimata, K., Klimstra, W. B., Bittman, R., Wilschut, J.
(2002). Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Viruses with Liposomes Containing Lipid-Conjugated Heparin. J. Virol.
76: 10128-10137
[Abstract]
[Full Text]
-
Lee, P., Knight, R., Smit, J. M., Wilschut, J., Griffin, D. E.
(2002). A Single Mutation in the E2 Glycoprotein Important for Neurovirulence Influences Binding of Sindbis Virus to Neuroblastoma Cells. J. Virol.
76: 6302-6310
[Abstract]
[Full Text]
-
Lee, E., Lobigs, M.
(2002). Mechanism of Virulence Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis Virus and Murray Valley Encephalitis Virus. J. Virol.
76: 4901-4911
[Abstract]
[Full Text]
-
Heil, M. L., Albee, A., Strauss, J. H., Kuhn, R. J.
(2001). An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety. J. Virol.
75: 6303-6309
[Abstract]
[Full Text]
-
Mandl, C. W., Kroschewski, H., Allison, S. L., Kofler, R., Holzmann, H., Meixner, T., Heinz, F. X.
(2001). Adaptation of Tick-Borne Encephalitis Virus to BHK-21 Cells Results in the Formation of Multiple Heparan Sulfate Binding Sites in the Envelope Protein and Attenuation In Vivo. J. Virol.
75: 5627-5637
[Abstract]
[Full Text]
-
Gardner, J. P., Frolov, I., Perri, S., Ji, Y., MacKichan, M. L., zur Megede, J., Chen, M., Belli, B. A., Driver, D. A., Sherrill, S., Greer, C. E., Otten, G. R., Barnett, S. W., Liu, M. A., Dubensky, T. W., Polo, J. M.
(2000). Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein. J. Virol.
74: 11849-11857
[Abstract]
[Full Text]
-
Lee, E., Lobigs, M.
(2000). Substitutions at the Putative Receptor-Binding Site of an Encephalitic Flavivirus Alter Virulence and Host Cell Tropism and Reveal a Role for Glycosaminoglycans in Entry. J. Virol.
74: 8867-8875
[Abstract]
[Full Text]
Top
Abstract
Introduction
