Previous Article | Next Article 
Journal of Virology, November 2000, p. 10508-10513, Vol. 74, No. 22
Department of Immunology/Microbiology,
Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois
606121; Department of Medical
Biochemistry and Microbiology, Section for Medical Biochemistry, The
Biomedical Center, SE-751 23 Uppsala,
Sweden2; and Laboratory of Infectious
Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland
20892-07203
Received 24 May 2000/Accepted 15 August 2000
Glycosaminoglycans (GAGs) on the surface of cultured cells are
important in the first step of efficient respiratory syncytial virus
(RSV) infection. We evaluated the importance of sulfation, the major
biosynthetic modification of GAGs, using an improved recombinant green
fluorescent protein-expressing RSV (rgRSV) to assay infection.
Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective
inhibitor of sulfation, for 48 h prior to inoculation reduced the
efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell
line deficient in N-sulfation was three times less
efficient than infection of the parental CHO cell line, indicating that
N-sulfation is important. In contrast, infection of a cell
line deficient in 2-O-sulfation was as efficient as
infection of the parental cell line, indicating that
2-O-sulfation is not required for RSV infection. Incubating
RSV with the purified soluble heparin, the prototype GAG, before
inoculation had previously been shown to neutralize its infectivity.
Here we tested chemically modified heparin chains that lack their
N-, C6-O-, or C2-O-sulfate groups.
Only heparin chains lacking the N-sulfate group lost the ability to neutralize infection, confirming that
N-sulfation, but not C6-O- or
C2-O-sulfation, is important for RSV infection. Analysis of
heparin fragments identified the 10-saccharide chain as the minimum
size that can neutralize RSV infectivity. Taken together, these results
show that, while sulfate modification is important for the ability of
GAGs to mediate RSV infection, only certain sulfate groups are
required. This specificity indicates that the role of cell surface GAGs
in RSV infection is not based on a simple charge interaction between
the virus and sulfate groups but instead involves a specific GAG
structural configuration that includes N-sulfate and a
minimum of 10 saccharide subunits. These elements, in addition to
iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264-275,
2000), partially define cell surface molecules important for RSV
infection of cultured cells.
Respiratory syncytial virus (RSV)
encodes 11 proteins, 3 of which are transmembrane surface glycoproteins
found in the viral envelope: the G (glycoprotein) (18, 40),
F (fusion) (11), and SH (small hydrophobic) (7,
12) proteins. The G protein is a type II transmembrane protein,
with an N-terminal transmembrane domain. The G protein is also found in
a smaller secreted form that lacks the transmembrane region, generated
by translation initiation at a second AUG in the mRNA and subsequent
proteolytic trimming (33). The G protein was identified as
the major viral attachment protein (27), but the isolation
of an infectious RSV mutant, cp-52, lacking its G and SH
genes (24), suggests that an attachment function can be
provided by the sole remaining glycoprotein, the F protein. However,
while the G protein appears to be dispensable for replication in vitro,
the highly attenuated nature of the cp-52 mutant in vivo
supports the idea that G protein is important for infection in vivo
(24). The classic role of the F protein is to initiate
infection by fusing the virion envelope with the target cell plasma
membrane. It is also responsible for cell-to-cell fusion. The role of
the SH protein is not clear. Its presence has been shown to enhance
fusion induced by the F protein when expressed from plasmids
(20). However, deleting the SH gene from recombinant virus
did not diminish its growth in cell culture (5) and
conferred only a small degree of attenuation in the respiratory tract
of chimpanzees (41).
Glycosaminoglycans (GAGs) are unbranched polysaccharide chains
associated with most mammalian cells. They are composed of repeating
disaccharide units of hexuronic acid and hexosamine. The hexuronic acid
is either D-glucuronate (GlcA) or its epimerized form,
L-iduronate (IdoA), and the hexosamine is either
N-acetylglucosamine (GlcNAc) or
N-acetylgalactosamine (GalNAc), depending on the type of
GAG. The GAG types that appear to be important for RSV infection in
HEp-2 cells are heparin sulfate (HS) and chondroitin sulfate B (CS-B)
(19). HS and CS-B are found on the surface of almost all
animal cells as components of proteoglycans. They are covalently linked
to the proteoglycan core proteins through O-glycosidic linkages to serine (44). Like most GAGs, HS and CS-B are
sulfated, though the level and position of the sulfate groups vary
among GAG types and even within the same GAG type in different tissues. The GAG heparin, which is found only in granulae of
connective-tissue-type mast cells, has a very similar structure to that
of HS but it is more heavily sulfated, especially at the N position of
GlcN, and it contains more IdoA. Heparin is frequently used as a
convenient analog of HS for experimental purposes (28), as
in the present study.
The biosynthesis of protein-bound GAGs such as HS and CS-B begins with
the transfer of xylose from UDP-xylose to the hydroxyl group of a
serine residue present in a serine-glycine motif in the core protein.
This is elongated into the tetrasaccharide, Many proteins bind heparin and other GAGs, including viral proteins,
enzymes, growth factors, and proteins of the extracellular matrix
(34). Binding is facilitated by the negative electrostatic charges on GAG chains provided by the sulfate groups and by the flexibility of IdoA, allowing positioning of the binding site. Many
viruses depend on cell surface GAG chains to efficiently initiate
infection of cultured cells. The first virus shown to require cell
surface HS was herpes simplex virus (HSV) (42). The group of
GAG-binding viruses has now expanded to include RSV (4, 19,
26), pseudorabies virus (38), Sindbis virus (6, 25), dengue virus (8), vaccinia virus (9,
22), adeno-associated virus type 2 (AAV2) (37), and
foot-and-mouth disease virus (23).
Previously we described a sensitive system to study the role of cell
surface GAGs in infection using recombinant green fluorescent protein
(GFP)-expressing (rgRSV). Cultured cells that were deficient in surface
GAGs due to mutation or enzymatic treatment were shown to be infected
with reduced efficiency. We also found that only those soluble GAGs
that contained IdoA were able to block infection (19).
Finally, a growth factor that specifically binds to cell surface IdoA
reduced the efficiency of RSV infection. These observations indicated
that efficient RSV infection requires cell surface GAGs and,
furthermore, that this requirement involved GAGs that contain IdoA,
specifically HS and CS-B. In the present study, we used an improved
version of rgRSV to further examine the role of GAGs, determining the
importance of their sulfate modifications in mediating RSV infection.
We found that N-sulfation of GAGs is particularly important
in rgRSV infection, that 2-O- and 6-O-sulfations
are not important, and that the minimal GAG length that is effective in
inhibiting rgRSV infection is 10 saccharides.
Cell lines and media.
The human epithelial cell line, HEp-2,
was maintained in OptiMEM (Life Technologies, Inc.) supplemented with
2% fetal bovine serum (FBS). CHO cell lines K1, pgsE-606
(3), and pgsF-17 (2) were provided by
Jeff Esko (University of California at San Diego) and maintained in
Dulbecco modified Eagle medium-F12 medium supplemented with 10% FBS.
Cells were incubated at 37°C in 5% CO2.
Preparation of an improved rgRSV.
The rgRSV(125) used in our
previous study (19) replicated in cell culture but produced
peak titers that were approximately 10-fold lower than its parent RSV.
In an attempt to enhance its replication, we modified the 3' terminus
of the full-length cDNA used to rescue rgRSV(125) so that it would
contain the first 75 nucleotides (nt) of the wild-type RSV rather than
only 54 nt. The additional 21 nt came from the untranslated region of
the native first gene, NS1. The new rgRSV(224) replicates more
efficiently, resulting in near-parental RSV titers and more rapid
development of fluorescence. The rgRSV(224) also produced syncytia at a
rate similar to the parental RSV and faster than rgRSV(125).
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Glycosaminoglycan Sulfation Requirements for
Respiratory Syncytial Virus Infection
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
GlcA-1,3
Gal-1,3Gal-1,4Xyl-1-O-Ser
(44). The nonreducing end of this tetrasaccharide
becomes a primer for GAG chain elongation. For example, heparin and HS
are synthesized as polymers of alternating GlcA and GlcNAc subunits.
Following GAG synthesis, some of the N-acetyl groups are
removed from the GlcNAc subunits and replaced with N-sulfate
groups. The N-deacetylation and N-sulfation
reactions are catalyzed by a single enzyme,
N-acetyltransferase (30, 39), and the sulfation
step involves the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (13). The N-sulfation reaction is required
for the next step: epimerization of some of the GlcA to IdoA at C5.
2-O-sulfation on the newly formed IdoA and
6-O-sulfation on GlcN are common. Less common sulfation
sites are at positions C3 of GlcN and C2 of GlcA. Sulfation at C3 of
GlcN is required for the formation of one of the functional receptors
for HSV (35). The biosynthesis of CS-B is similar, except
that the hexosamine is GalNAc instead of GlcNAc and there are
differences in the postsynthetic modifications of the polysaccharide chain.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Chemicals. The following chemicals were purchased from the Sigma Chemical Company: dextran from Leuconostoc mesenteroides, average molecular mass of ~10 kDa (D-9260); dextran sulfate, 5 kDa (D-7037); dextran sulfate, 10 kDa (D-6924), D-glucosamine 2-sulfate (G-7889); D-glucosamine 6-sulfate (G-8641); D-glucosamine 3,4,6-trisulfate (G-5533); D-glucosamine 3-sulfate (G-4267); D-glucosamine 2,6-disulfate (G-7514); D-glucosamine 2,3-disulfate (G-7639); D-glucosamine N2,3,6-trisulfate, and heparin disaccharide III-S (H-9392). Neoparin, Inc. (San Leandro, Calif.), was the commercial supplier for bovine intestinal heparin as well as derivatives that had been chemically modified: N-desulfated, fully N-sulfated, 6-O-desulfated, or 2-O-desulfated.
Fragments of bovine lung heparin oligosaccharides of 4-, 6-, 10-, 14-, 16-, 18- to 20-, and 22-mer lengths were prepared by limited deaminative cleavage at pH 1.5 and size fractionated as described previously (17, 36).Neutralization assays. Soluble molecules were diluted twofold serially in serum-free OptiMEM medium. An equal volume of rgRSV was added to each dilution, and the mixtures were incubated 45 min at 20°C to allow binding. Mixtures were then used to inoculate 24-h-old HEp-2 cell monolayers in 12-well plates, with periodic agitation. The inoculum was added at a multiplicity of infection (MOI) of 1 to 2 PFU per cell (PFU was determined on HEp-2 cells). Unbound virus was removed after 2 h, and cells were washed once with phosphate-buffered saline (PBS). Complete medium was added to cells, and they were incubated for an additional 24 h. Cells were then trypsinized by 1× Trypsin-EDTA (Gibco-BRL), fixed with 2% paraformaldehyde, and analyzed by flow cytometry to detect GFP-expressing cells as described elsewhere (19).
Infection of HEp-2 cells under conditions of sulfate depletion. HEp-2 cells were seeded on six-well plates in OptiMEM-2% FBS and incubated overnight to form monolayers. They were then washed twice with PBS and incubated in sulfate-free medium, Joklik-modified S-MEM, (Gibco-BRL catalog no. 22300) supplemented with 10% dialyzed FBS (Gibco-BRL catalog no. 26300) in the presence or absence of 50 mM sodium chlorate (Aldrich catalog no. 403016), or magnesium sulfate as a control to replenish sulfate. After 24 h, cells were washed with PBS and inoculated with rgRSV as described above. After a 2-h adsorption period, the cells were washed with complete medium and incubated for 24 h in complete medium. Cells were then processed for flow cytometry as described above. In addition, an aliquot of each harvested monolayer was assayed for cell number.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Effect of dextran sulfate on rgRSV infection. Dextran, a bacterial product, is a branched glucose polysaccharide that can be chemically sulfated to generate dextran sulfate. Up to three sulfate groups modify each glucose unit of dextran sulfate, composing 17% of its weight. Dextran sulfate has been shown to neutralize several viruses, including RSV (21).
To confirm this finding and to determine the contribution of sulfate and dextran size to this neutralizing effect, we treated rgRSV with unsulfated dextran (average size, 10 kDa) and with dextran sulfate (average size, 5 or 10 kDa). Both sulfated dextrans neutralized rgRSV in a dose-dependent manner, and neutralization was more efficient with the larger polysaccharide (Fig. 1). In contrast, unsulfated dextran was completely inactive in neutralizing rgRSV. These findings confirmed that dextran sulfate can neutralize RSV infectivity very efficiently but showed that this activity was completely dependent on the presence of sulfate groups.
|
Treatment with sodium chlorate reduces the susceptibility of cells
to rgRSV infection.
Sodium chlorate acts as a sulfate analog,
replacing the sulfate group in the sulfate donor for GAG synthesis,
PAPS (14). The resulting
3'-phosphoadenosine-5'-phosphochlorate donates unstable chlorate groups
to GAG chains that are spontaneously hydrolyzed (1). If GAG
sulfation were critical for RSV infection, then sodium chlorate
treatment of HEp-2 cells would be predicted to inhibit subsequent rgRSV
infection. To test this possibility, cells were preincubated in
sulfate-free medium to reduce the availability of sulfate (Fig.
2, sample 2). A second set was
preincubated in sulfate-free medium containing 50 mM sodium chlorate to
inhibit the incorporation of sulfate into cellular GAGs (sample 3). A third set of cells was incubated in sulfate-free medium in which the
sulfate was replenished by the addition of MgSO4 (sample
1). Following a preincubation of 48 h, the cells were inoculated
with rgRSV, incubated for an additional 24 h in complete medium,
trypsinized, fixed, and analyzed for the expression of GFP. The
efficiency of rgRSV infection in cells treated with chlorate was 40%
that of control cells incubated in medium containing MgSO4
(compare sample 3 with sample 1). These results supported the idea that sulfation is important for efficient rgRSV infection.
|
Sensitivity of sulfation-deficient CHO cell lines to rgRSV
infection.
The most common positions of sulfate addition in
heparin, HS, and CS-B are the N and C6 positions of GlcN and the C2
position of IdoA. To determine whether N-sulfation of GAGs
is required for efficient RSV infection, we tested the sensitivity of
the CHO pgsE-606 cell line to infection with rgRSV. This
mutant cell line expresses normal levels of GAGs but has three- to
fivefold less N-acetyltransferase activity (3).
As a result, its GAGs are undersulfated on GlcN. These mutant cells and
their CHO K1 parental cells were inoculated with rgRSV and incubated
for 36 h at 37°C, after which infected cells were counted by
flow cytometry. CHO pgsE-606 cells were threefold less
sensitive to infection than the parental K1 cells (Fig.
3), indicating that
N-sulfation is important for efficient infection. However,
since the formation of IdoA in heparan sulfate requires the presence of
N-sulfate on GlcN (Introduction), it was not clear from this
result alone whether the reduction of infection in this experiment was
directly due to the absence of N-sulfate groups or was a
consequence of a lower content of IdoA in the cell surface GAGs. This
point is addressed below.
|
Effects of soluble, chemically modified heparin on rgRSV
infection.
As mentioned above, preincubation of RSV with soluble
heparin reduces the infectivity of the virus, presumably by saturating GAG-binding sites needed for efficient infection. To further test the
importance of GAG sulfation, especially that of N-sulfation, we compared the neutralization activity of unmodified soluble heparin
with that of derivatives that had been modified chemically as follows:
(i) to remove N-sulfate groups, (ii) to remove the 6-O-sulfate groups, (iii) to remove the
2-O-sulfate groups, or (iv) to add N-sulfate
groups to all GlcNAc subunits. These heparins were serially diluted,
and each dilution was incubated with rgRSV before inoculating the HEp-2
cells. Unmodified heparin efficiently inhibited rgRSV infection (50%
inhibitory concentration [IC50] of 6 µg/ml), a result
consistent with results in previous studies (4, 19, 26).
Heparin that had been treated to add N-sulfates to all
remaining unmodified GlcN residues was not significantly different from
the native form (Fig. 4), which was not surprising given the high
degree of N-sulfation of native heparin. In contrast, heparin lacking N-sulfation inhibited infection very poorly
(IC50 of >200 µg/ml). The
neutralizing ability of heparin, therefore, appears to be highly
dependent on N-sulfation. Conversely, both 6-O-desulfated and 2-O-desulfated heparin
retained nearly their full inhibitory activity, indicating that sulfate
at position C6 of GlcN or position C2 of GlcA or IdoA are not required
for efficient binding by rgRSV.
|
Importance of heparin chain length in rgRSV neutralization.
To
further define the GAG structural features that are important for
binding to RSV, we examined the effect of chain length on the ability
of heparin to neutralize rgRSV. This analysis first examined the free
monosaccharides GlcA and GlcN. Neither monosaccharide significantly
inhibited rgRSV infection (Table 1). We
also tested glucosamine monosaccharides with sulfate groups at
positions 2, 3, 2,3, 2,6, and N2,3,6. None of
these sugars inhibited rgRSV infection even at concentrations up to 400 µg/ml (Table 1).
|
HexA2S1-4GlcNS (III-S), resulting from the digestion of
heparin or HS with heparinases I and II. Like the monosaccharides, it
was unable to inhibit rgRSV infection at the tested concentrations. These results suggested that the ability to bind to and neutralize RSV
depended on a larger GAG size and/or additional structural components
found on heparin chains.
We then tested bovine lung heparin fragments of increasing length,
namely, 4-, 6-, 8-, 10-, 14-, 16-, 18- to 20-, and 22-mer saccharide
subunits, for the ability to neutralize rgRSV infection (Fig.
5). Even at the highest concentration
tested (200 µg/ml), the 4-, 6-, and 8-mer fragments did not
significantly neutralize rgRSV infectivity. The minimum chain length
that provided detectable neutralization of rgRSV was a 10-mer, although
inhibition was relatively weak (IC50 of >200 µg/ml).
Inhibition of infection increased with increasing heparin chain length,
with a mixture of 22-mer and longer chains neutralizing rgRSV as
efficiently as native heparin.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We previously showed that HS and CS-B, two GAGs that are found on cell surface proteoglycans, are important for efficient RSV infection (19). HS, CS-B, and heparin, the latter being a structural analog of HS, were each able to neutralize rgRSV infectivity when preincubated with the virus. All three of these GAGs contain IdoA, whereas three other GAGs that do not contain IdoA did not neutralize rgRSV. In the present study, the presence of sulfate groups was found to be important for polysaccharide binding to RSV and for RSV infection of cultured cells. Furthermore, we examined the importance of sulfation at specific GAG positions by using mutant cell lines that are defective in specific sulfotransferases and by using soluble heparin and modified heparins.
Dextran sulfate strongly inhibited rgRSV infection, as is evident from the IC50 of 0.1 µg/ml for 10K dextran sulfate, compared to that of heparin (1.0 µg/ml), HS (12.5 µg/ml), and CS-B (200 µg/ml) (19). The very strong binding of dextran sulfate indicated by these data is dependent on its heavy sulfation, since desulfated dextran sulfate was completely inactive in neutralizing rgRSV. Although the very high sulfate content and branched nature of dextran sulfate makes it an inexact model for cell surface GAGs such as HS and CS-B, the strong inhibition observed shows that the binding of RSV to sulfated polysaccharides can be very strong indeed and that sulfation is important.
Chlorate treatment of cells reduces the overall level of GAG sulfation and has been shown to reduce infection by vaccinia virus (9) and human immunodeficiency virus type 1 (31), but not by AAV2 (32), despite the fact that HS is thought to act as a receptor for AAV2 (37). In our experiments, sodium chlorate pretreatment of cells reduced the efficiency of rgRSV infection to 40% that of untreated cells, suggesting a role for sulfation.
Most of the GlcN residues in heparin and approximately half of the GlcN residues in HS (29) are modified by substitution of their N-acetyl groups with N-sulfate, generating N-sulfated GlcN. There are other sulfation sites in heparin and HS, most notably the C6 position of GlcN and the C2 position of IdoA. Infrequent sulfation also occurs at the C3 position of GlcN and the C2 position of GlcA (28). Here we provided evidence that N-sulfation, but not C6 or C2 sulfation, is required for efficient RSV infection. This conclusion is supported by two observations. (i) The pgsE-606 CHO-cell line, deficient in N-sulfation but expressing normal levels of cell surface HS and CS, was only 34% as permissive to rgRSV infection as the parent cell line. This reduction in virus infectivity is similar in magnitude to the decrease (three- to fivefold) in N-sulfation enzymatic activity (3). On the other hand, CHO pgsF-17, a cell line deficient in C2-O-sulfation, was fully permissive for rgRSV infection. (ii) Chemically modified, full-length N-desulfated heparin lost its neutralizing activity. These results indicate that the N-sulfation is important in rgRSV binding. Both 6-O-desulfated and 2-O-desulfated heparin chains retained their neutralizing activity, indicating that these sulfate groups are not important for binding to rgRSV.
The conclusion most consistent with these data is that particular sulfate groups on GAGs, rather than the total amount of sulfate, are important for promoting rgRSV infection. Besides the involvement of N-sulfate groups, polyvalence appears to be important. Neither highly sulfated mono- or disaccharides nor heparin fragments shorter than 10-mer were effective in neutralizing rgRSV. This indicates that there is a need for longer ligands, either due to specific features within the binding site or a requirement of polyvalent interaction with virion surface components.
The finding that efficient binding of RSV to GAGs requires IdoA, N-sulfation, and a minimum saccharide chain length of 10 indicates that the binding is not simply a function of charge but instead has greater specificity. In the case of dengue virus, a 10-saccharide heparin fragment was required to inhibit virus binding to host cells (8), whereas HSV was inhibited by a 12-saccharide fragment (17). This 12-mer must contain at least one 2-O- and one 6-O-sulfate group, and yet no N-sulfate is necessary. This difference between the HSV specificity and the RSV requirement for only N-sulfation demonstrated in the present here clearly indicates that different viruses have different GAG-binding specificities. These viruses have subtle but distinct differences in the specificities of their interaction with cell surface GAGs.
While it is clear that cell surface GAGs are required for efficient rgRSV infection of cultured cells, it is not clear whether this binding represents attachment in toto, as appears to be the case for AAV2 (37), or whether the interaction between RSV and GAGs represents a low-affinity first step in attachment followed by a second step that remains to be identified. This latter case would resemble the situation with HSV, where binding to GAGs is an initial step that enhances the ability of the virus to find its specific receptor (42). It is also unclear which of the three RSV glycoproteins is responsible for binding to GAGs to initiate infection. Synthetic peptides representing the consensus regions in the G protein of subgroups A and B of RSV were found to bind to Vero cells and inhibit RSV infection. Heparin prevented this peptide from binding to Vero cells (16), suggesting that this segment of the G protein binds to cell surface GAGs. But the ability of the RSV mutant cp-52 to infect cultured cells, even though it lacks the genes for both the G and SH proteins (24), would suggest that the F protein can also function as an attachment protein.
Recently, Feldman et al. (15) reported that the F protein released from RSV-infected cells by detergent lysis also binds to heparin, that cp-52 is neutralized by heparin, and that pretreatment of cells with heparinase inhibits cp-52 infection. We have made similar observations with rgRSV lacking both the SH and G genes (S. Techaarpornkul, N. Barretto, P. L. Collins, and M. E. Peeples, manuscript in preparation). These results suggest that the F protein also binds to GAGs on the cell surface to initiate infection in the absence of the G protein. It is not yet clear whether the GAG-binding specificities of the F and G proteins are identical; what the relative contributions of the F, G, and SH proteins are to initiating infection; or whether these proteins act cooperatively.
| |
ACKNOWLEDGMENTS |
|---|
We thank Greg Spear and Alan Landay for use of the flow cytometer, Jeff Esko and Patricia Spear for providing the CHO cell lines, Ada Cole for use of the inverted fluorescence microscope, and Barbara Newton for technical support.
This work was supported by a grant from the Rush University Committee on Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612. Phone: (312) 942-8736. Fax: (312) 942-2808. E-mail: mpeeples{at}rush.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Baeuerle, P. A., and W. B. Huttner. 1986. Chlorate: a potent inhibitor of protein sulfation in intact cells. Biochem. Biophys. Res. Commun. 141:870-877[CrossRef][Medline]. |
| 2. |
Bai, X., and J. D. Esko.
1996.
An animal cell mutant defective in heparan sulfate hexuronic acid 2-O-sulfation.
J. Biol. Chem.
271:17711-17717 |
| 3. |
Bame, K. J., and J. D. Esko.
1989.
Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase.
J. Biol. Chem.
264:8059-8065 |
| 4. |
Bourgeois, C.,
J. B. Bour,
K. Lidholt,
C. Gauthray, and P. Pothier.
1998.
Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro.
J. Virol.
72:7221-7227 |
| 5. | Bukreyev, A., S. S. Whitehead, B. R. Murphy, and P. L. Collins. 1997. Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J. Virol. 71:8973-8982[Abstract]. |
| 6. |
Byrnes, A. P., and D. E. Griffin.
1998.
Binding of Sindbis virus to cell surface heparan sulfate.
J. Virol.
72:7349-7356 |
| 7. |
Cane, P. A., and C. R. Pringle.
1991.
Respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (SH gene) and restriction mapping (N gene).
J. Gen. Virol.
72:349-357 |
| 8. | 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]. |
| 9. |
Chung, C. S.,
J. C. Hsiao,
Y. S. Chang, and W. Chang.
1998.
A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate.
J. Virol.
72:1577-1585 |
| 10. |
Collins, P. L.,
M. G. Hill,
E. Camargo,
H. Grosfeld,
R. M. Chanock, and B. R. Murphy.
1995.
Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.
Proc. Natl. Acad. Sci. USA
92:11563-11567 |
| 11. |
Collins, P. L.,
Y. T. Huang, and G. W. Wertz.
1984.
Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus.
Proc. Natl. Acad. Sci. USA
81:7683-7687 |
| 12. |
Collins, P. L.,
R. A. Olmsted, and P. R. Johnson.
1990.
The small hydrophobic protein of human respiratory syncytial virus: comparison between antigenic subgroups A and B.
J. Gen. Virol.
71:1571-1576 |
| 13. | Conard, H. E. 1998. Structures of heparinoids, p. 7-60. In Heparin binding proteins. Academic Press, San Diego, Calif. |
| 14. | Conard, H. E. 1998. The cellular metabolism of heparan sulfate, p. 137-182. In Heparin binding proteins. Academic Press, San Diego, Calif. |
| 15. |
Feldman, S. A.,
S. Audet, and J. A. Beeler.
2000.
The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.
J. Virol.
74:6442-6447 |
| 16. |
Feldman, S. A.,
R. M. Hendry, and J. A. Beeler.
1999.
Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G.
J. Virol.
73:6610-6617 |
| 17. |
Feyzi, E.,
E. Trybala,
T. Bergstrom,
U. Lindahl, and D. Spillmann.
1997.
Structural requirement of heparan sulfate for interaction with herpes simplex virus type 1 virions and isolated glycoprotein C.
J. Biol. Chem.
272:24850-24857 |
| 18. |
Gruber, C., and S. Levine.
1983.
Respiratory syncytial virus polypeptides. III. The envelope-associated proteins.
J. Gen. Virol.
64:825-832 |
| 19. | Hallak, L. K., P. L. Collins, W. Knudson, and M. E. Peeples. 2000. Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. Virology 271:264-275[CrossRef][Medline]. |
| 20. | Heminway, B. R., Y. Yu, Y. Tanaka, K. G. Perrine, E. Gustafson, J. M. Bernstein, and M. S. Galinski. 1994. Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology 200:801-805[CrossRef][Medline]. |
| 21. |
Hosoya, M.,
J. Balzarini,
S. Shigeta, and E. De Clercq.
1991.
Differential inhibitory effects of sulfated polysaccharides and polymers on the replication of various myxoviruses and retroviruses, depending on the composition of the target amino acid sequences of the viral envelope glycoproteins.
Antimicrob. Agents Chemother.
35:2515-2520 |
| 22. |
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 |
| 23. |
Jackson, T.,
F. M. Ellard,
R. A. Ghazaleh,
S. M. Brookes,
W. E. Blakemore,
A. H. Corteyn,
D. I. Stuart,
J. W. Newman, and A. M. 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 |
| 24. |
Karron, R. A.,
D. A. Buonagurio,
A. F. Georgiu,
S. S. Whitehead,
J. E. Adamus,
M. L. Clements-Mann,
D. O. Harris,
V. B. Randolph,
S. A. Udem,
B. R. Murphy, and M. S. Sidhu.
1997.
Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant.
Proc. Natl. Acad. Sci. USA
94:13961-13966 |
| 25. |
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 |
| 26. | 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]. |
| 27. |
Levine, S.,
R. Klaiber-Franco, and P. R. Paradiso.
1987.
Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus.
J. Gen. Virol.
68:2521-2524 |
| 28. | Lindahl, U., K. Lidholt, D. Spillmann, and L. Kjellen. 1994. More to "heparin" than anticoagulation. Thromb. Res. 75:1-32[CrossRef][Medline]. |
| 29. |
Maccarana, M.,
Y. Sakura,
A. Tawada,
K. Yoshida, and U. Lindahl.
1996.
Domain structure of heparan sulfates from bovine organs.
J. Biol. Chem.
271:17804-17810 |
| 30. |
Mandon, E.,
E. S. Kempner,
M. Ishihara, and C. B. Hirschberg.
1994.
A monomeric protein in the Golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate.
J. Biol. Chem.
269:11729-11733 |
| 31. | Ohshiro, Y., T. Murakami, K. Matsuda, K. Nishioka, K. Yoshida, and N. Yamamoto. 1996. Role of cell surface glycosaminoglycans of human T cells in human immunodeficiency virus type-1 (HIV-1) infection. Microbiol. Immunol. 40:827-835[Medline]. |
| 32. | Qiu, J., A. Handa, M. Kirby, and K. E. Brown. 2000. The interaction of heparin sulfate and adeno-associated virus 2. Virology 269:137-147[CrossRef][Medline]. |
| 33. |
Roberts, S. R.,
D. Lichtenstein,
L. A. Ball, and G. W. Wertz.
1994.
The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons.
J. Virol.
68:4538-4546 |
| 34. | Salmivirta, M., K. Lidholt, and U. Lindahl. 1996. Heparan sulfate: a piece of information. FASEB J. 10:1270-1279[Abstract]. |
| 35. | Shukla, D., J. Liu, P. Blaiklock, N. W. Shworak, X. Bai, J. D. Esko, G. H. Cohen, R. J. Eisenberg, R. D. Rosenberg, and P. G. Spear. 1999. A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99:13-22[CrossRef][Medline]. |
| 36. |
Spillmann, D.,
D. Witt, and U. Lindahl.
1998.
Defining the interleukin-8-binding domain of heparan sulfate.
J. Biol. Chem.
273:15487-15493 |
| 37. |
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 |
| 37a. |
Teng, M. N., and P. L. Collins.
1998.
Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles.
J Virol.
72:5707-5716 |
| 38. | Voigt, A., D. Sawitzky, H. Zeichhardt, and K. O. Habermehl. 1995. Cellular receptor structures for pseudorabies virus are blocked by antithrombin III. Med. Microbiol. Immunol. 184:97-103[Medline]. |
| 39. |
Wei, Z.,
S. J. Swiedler,
M. Ishihara,
A. Orellana, and C. B. Hirschberg.
1993.
A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate.
Proc. Natl. Acad. Sci. USA
90:3885-3888 |
| 40. |
Wertz, G. W.,
P. L. Collins,
Y. Huang,
C. Gruber,
S. Levine, and L. A. Ball.
1985.
Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein.
Proc. Natl. Acad. Sci. USA
82:4075-4079 |
| 41. |
Whitehead, S. S.,
A. Bukreyev,
M. N. Teng,
C. Y. Firestone,
M. St. Claire,
W. R. Elkins,
P. L. Collins, and B. R. Murphy.
1999.
Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees.
J. Virol.
73:3438-3442 |
| 42. |
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 |
| 43. | Wyatt, L. S., B. Moss, and S. Rozenblatt. 1995. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology 210:202-205[CrossRef][Medline]. |
| 44. |
Zhang, L., and J. D. Esko.
1994.
Amino acid determinants that drive heparan sulfate assembly in a proteoglycan.
J. Biol. Chem.
269:19295-19299 |
Journal of Virology, November 2000, p. 10508-10513, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kwilas, S., Liesman, R. M., Zhang, L., Walsh, E., Pickles, R. J., Peeples, M. E.
(2009). Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans. J. Virol.
83: 10710-10718
[Abstract] [Full Text] -
Dyer, K. D., Percopo, C. M., Fischer, E. R., Gabryszewski, S. J., Rosenberg, H. F.
(2009). Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6. Blood
114: 2649-2656
[Abstract] [Full Text] -
Groskreutz, D. J., Monick, M. M., Babor, E. C., Nyunoya, T., Varga, S. M., Look, D. C., Hunninghake, G. W.
(2009). Cigarette Smoke Alters Respiratory Syncytial Virus-Induced Apoptosis and Replication. Am. J. Respir. Cell Mol. Bio.
41: 189-198
[Abstract] [Full Text] -
de Graaf, M., Schrauwen, E. J. A., Herfst, S., van Amerongen, G., Osterhaus, A. D. M. E., Fouchier, R. A. M.
(2009). Fusion protein is the main determinant of metapneumovirus host tropism. J. Gen. Virol.
90: 1408-1416
[Abstract] [Full Text] -
Wang, H, Su, Z, Schwarze, J
(2009). Healthy but not RSV-infected lung epithelial cells profoundly inhibit T cell activation. Thorax
64: 283-290
[Abstract] [Full Text] -
Thammawat, S., Sadlon, T. A., Hallsworth, P. G., Gordon, D. L.
(2008). Role of Cellular Glycosaminoglycans and Charged Regions of Viral G Protein in Human Metapneumovirus Infection. J. Virol.
82: 11767-11774
[Abstract] [Full Text] -
Utley, T. J., Ducharme, N. A., Varthakavi, V., Shepherd, B. E., Santangelo, P. J., Lindquist, M. E., Goldenring, J. R., Crowe, J. E. Jr.
(2008). Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2. Proc. Natl. Acad. Sci. USA
105: 10209-10214
[Abstract] [Full Text] -
Collins, P. L., Graham, B. S.
(2008). Viral and Host Factors in Human Respiratory Syncytial Virus Pathogenesis. J. Virol.
82: 2040-2055
[Full Text] -
Chapman, J., Abbott, E., Alber, D. G., Baxter, R. C., Bithell, S. K., Henderson, E. A., Carter, M. C., Chambers, P., Chubb, A., Cockerill, G. S., Collins, P. L., Dowdell, V. C. L., Keegan, S. J., Kelsey, R. D., Lockyer, M. J., Luongo, C., Najarro, P., Pickles, R. J., Simmonds, M., Taylor, D., Tyms, S., Wilson, L. J., Powell, K. L.
(2007). RSV604, a Novel Inhibitor of Respiratory Syncytial Virus Replication. Antimicrob. Agents Chemother.
51: 3346-3353
[Abstract] [Full Text] -
Kolokoltsov, A. A., Deniger, D., Fleming, E. H., Roberts, N. J. Jr., Karpilow, J. M., Davey, R. A.
(2007). Small Interfering RNA Profiling Reveals Key Role of Clathrin-Mediated Endocytosis and Early Endosome Formation for Infection by Respiratory Syncytial Virus. J. Virol.
81: 7786-7800
[Abstract] [Full Text] -
Bitko, V., Shulyayeva, O., Mazumder, B., Musiyenko, A., Ramaswamy, M., Look, D. C., Barik, S.
(2007). Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-{kappa}B-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth. J. Virol.
81: 1786-1795
[Abstract] [Full Text] -
Crim, R. L., Audet, S. A., Feldman, S. A., Mostowski, H. S., Beeler, J. A.
(2007). Identification of Linear Heparin-Binding Peptides Derived from Human Respiratory Syncytial Virus Fusion Glycoprotein That Inhibit Infectivity. J. Virol.
81: 261-271
[Abstract] [Full Text] -
Moore, P. E., Cunningham, G., Calder, M. M., DeMatteo, A. D. Jr., Peeples, M. E., Summar, M. L., Peebles, R. S. Jr.
(2006). Respiratory Syncytial Virus Infection Reduces beta2-Adrenergic Responses in Human Airway Smooth Muscle. Am. J. Respir. Cell Mol. Bio.
35: 559-564
[Abstract] [Full Text] -
Barth, H., Schnober, E. K., Zhang, F., Linhardt, R. J., Depla, E., Boson, B., Cosset, F.-L., Patel, A. H., Blum, H. E., Baumert, T. F.
(2006). Viral and Cellular Determinants of the Hepatitis C Virus Envelope-Heparan Sulfate Interaction. J. Virol.
80: 10579-10590
[Abstract] [Full Text] -
Chi, B., Dickensheets, H. L., Spann, K. M., Alston, M. A., Luongo, C., Dumoutier, L., Huang, J., Renauld, J.-C., Kotenko, S. V., Roederer, M., Beeler, J. A., Donnelly, R. P., Collins, P. L., Rabin, R. L.
(2006). Alpha and lambda interferon together mediate suppression of CD4 T cells induced by respiratory syncytial virus.. J. Virol.
80: 5032-5040
[Abstract] [Full Text] -
Guerrero-Plata, A., Casola, A., Suarez, G., Yu, X., Spetch, L., Peeples, M. E., Garofalo, R. P.
(2006). Differential Response of Dendritic Cells to Human Metapneumovirus and Respiratory Syncytial Virus. Am. J. Respir. Cell Mol. Bio.
34: 320-329
[Abstract] [Full Text] -
Vlasak, M., Goesler, I., Blaas, D.
(2005). Human Rhinovirus Type 89 Variants Use Heparan Sulfate Proteoglycan for Cell Attachment. J. Virol.
79: 5963-5970
[Abstract] [Full Text] -
Zhang, L., Bukreyev, A., Thompson, C. I., Watson, B., Peeples, M. E., Collins, P. L., Pickles, R. J.
(2005). Infection of Ciliated Cells by Human Parainfluenza Virus Type 3 in an In Vitro Model of Human Airway Epithelium. J. Virol.
79: 1113-1124
[Abstract] [Full Text] -
Fayzulin, R., Gorchakov, R., Petrakova, O., Volkova, E., Frolov, I.
(2005). Sindbis Virus with a Tricomponent Genome. J. Virol.
79: 637-643
[Abstract] [Full Text] -
San-Juan-Vergara, H., Peeples, M. E., Lockey, R. F., Mohapatra, S. S.
(2004). Protein Kinase C-{alpha} Activity Is Required for Respiratory Syncytial Virus Fusion to Human Bronchial Epithelial Cells. J. Virol.
78: 13717-13726
[Abstract] [Full Text] -
Budge, P. J., Graham, B. S.
(2004). Inhibition of respiratory syncytial virus by RhoA-derived peptides: implications for the development of improved antiviral agents targeting heparin-binding viruses. J Antimicrob Chemother
54: 299-302
[Abstract] [Full Text] -
Elliott, M. B., Pryharski, K. S., Yu, Q., Parks, C. L., Laughlin, T. S., Gupta, C. K., Lerch, R. A., Randolph, V. B., LaPierre, N. A., Dack, K. M. H., Hancock, G. E.
(2004). Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro. J. Virol.
78: 5773-5783
[Abstract] [Full Text] -
Budge, P. J., Li, Y., Beeler, J. A., Graham, B. S.
(2004). RhoA-Derived Peptide Dimers Share Mechanistic Properties with Other Polyanionic Inhibitors of Respiratory Syncytial Virus (RSV), Including Disruption of Viral Attachment and Dependence on RSV G. J. Virol.
78: 5015-5022
[Abstract] [Full Text] -
Easton, A. J., Domachowske, J. B., Rosenberg, H. F.
(2004). Animal Pneumoviruses: Molecular Genetics and Pathogenesis. Clin. Microbiol. Rev.
17: 390-412
[Abstract] [Full Text] -
Escribano-Romero, E., Rawling, J., Garcia-Barreno, B., Melero, J. A.
(2004). The Soluble Form of Human Respiratory Syncytial Virus Attachment Protein Differs from the Membrane-Bound Form in Its Oligomeric State but Is Still Capable of Binding to Cell Surface Proteoglycans. J. Virol.
78: 3524-3532
[Abstract] [Full Text] -
Zhang, Y., Rassa, J. C., deObaldia, M. E., Albritton, L. M., Ross, S. R.
(2003). Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein. J. Virol.
77: 10468-10478
[Abstract] [Full Text] -
Douglas, J. L., Panis, M. L., Ho, E., Lin, K.-Y., Krawczyk, S. H., Grant, D. M., Cai, R., Swaminathan, S., Cihlar, T.
(2003). Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein. J. Virol.
77: 5054-5064
[Abstract] [Full Text] -
Schlender, J., Zimmer, G., Herrler, G., Conzelmann, K.-K.
(2003). Respiratory Syncytial Virus (RSV) Fusion Protein Subunit F2, Not Attachment Protein G, Determines the Specificity of RSV Infection. J. Virol.
77: 4609-4616
[Abstract] [Full Text] -
Zimmer, G., Conzelmann, K.-K., Herrler, G.
(2002). Cleavage at the Furin Consensus Sequence RAR/KR109 and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture. J. Virol.
76: 9218-9224
[Abstract] [Full Text] -
Zhang, L., Peeples, M. E., Boucher, R. C., Collins, P. L., Pickles, R. J.
(2002). Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology. J. Virol.
76: 5654-5666
[Abstract] [Full Text] -
Gorman, J. J., McKimm-Breschkin, J. L., Norton, R. S., Barnham, K. J.
(2001). Antiviral Activity and Structural Characteristics of the Nonglycosylated Central Subdomain of Human Respiratory Syncytial Virus Attachment (G) Glycoprotein. J. Biol. Chem.
276: 38988-38994
[Abstract] [Full Text] -
Hurrelbrink, R. J., McMinn, P. C.
(2001). Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein. J. Virol.
75: 7692-7702
[Abstract] [Full Text] -
Techaarpornkul, S., Barretto, N., Peeples, M. E.
(2001). Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene. J. Virol.
75: 6825-6834
[Abstract] [Full Text] -
Zimmer, G., Trotz, I., Herrler, G.
(2001). N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus. J. Virol.
75: 4744-4751
[Abstract] [Full Text] -
Zimmer, G., Budz, L., Herrler, G.
(2001). Proteolytic Activation of Respiratory Syncytial Virus Fusion Protein. CLEAVAGE AT TWO FURIN CONSENSUS SEQUENCES. J. Biol. Chem.
276: 31642-31650
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»