Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, S-413 46 Göteborg, Sweden1
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC, CanadaV6T 1Z32
Author for correspondence: Kristina Mårdberg. Fax +46 31 82 70 32. e-mail Kristina.Mardberg{at}microbio.gu.se
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Abstract |
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Introduction |
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In HSV-1, binding to cell surface HS during attachment is mediated primarily by glycoprotein C (gC), in which a functional domain responsible for this interaction has been defined (Herold et al., 1991 ; M
rdberg et al., 2001
; Svennerholm et al., 1991
; Tal-Singer et al., 1995
; Trybala et al., 1994
). Under some conditions, HSV-1 may utilize chondroitin sulfate (CS) for attachment to the cell surface (Banfield et al., 1995b
), but the HSV glycoprotein responsible for CS binding has not been identified. In addition, it has been reported that CS may serve as a receptor for some other viruses (Bruett et al., 2000
; Hsiao et al., 1999
). The CS molecule is, in parallel with HS, a GAG that is found in many tissues in the human body, including the epidermis and connecting basal membrane of the human skin, where HSV-1 causes tissue damage during symptomatic reactivation (Huff et al., 1981
; Murdoch et al., 1994
; Sorrell et al., 1999
; Zimmermann et al., 1994
). In polarized MDCK cells, which express CS predominately on their apical surfaces (Kolset et al., 1999
), HSV-1 infection by this route was shown to be gC-dependent (Sears et al., 1991
). These two families of GAG molecules show several similar features, such as the formation of long saccharide chains that are modified extensively by sulfation at selected positions and exposure into the extracellular matrix. It is therefore of interest to investigate features of HSV-1 interaction with HS and CS under comparable conditions.
Using HSV-1, mutant gro2C cells exposing only CS on the cell surface and variants deficient in HS and CS expression were sequentially derived from mouse fibroblast L cells, as reported earlier (Banfield et al., 1995a ; Gruenheid et al., 1993
). In a virus-resistant cell line lacking both CS and HS expression (sog9 cells), permissiveness to HSV-1 infection was restored by transfection of the EXT-1 gene from HeLa cells, enabling a regained capacity to synthesize HS (McCormick et al., 1998
). This gave us the opportunity to compare the role of gC and its functional domain during virus entry in cells sharing the same ancestry but expressing both HS and CS, either HS or CS or lacking the sulfated GAG molecules altogether. Using a panel of HSV-1 strains carrying point-mutations in gC, a gC-positive (gC+) and a gC-negative (gC-) HSV-1 strain (M
rdberg et al., 2001
), we found that attachment to and infection of a CS-expressing cell line (gro2C cells) was gC-dependent and that binding to these cells was mediated by a gC domain similar to that utilized for virus attachment to HS-exposing sog9 EXT-1 cells. Furthermore, the gCCS and gCHS interactions exhibited subtle but distinct differences, suggesting a flexibility of the viral gC protein in binding to different forms of cell surface GAG molecules.
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Methods |
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Purification of extracellular virus.
GMK-AH1 cells were infected with mutant viruses, KOS321 or gC-39, at an m.o.i. of 3 p.f.u. per cell. Following virus adsorption for 12 h at 37 °C, cells were washed twice with EMEM and 45 ml of fresh EMEM supplemented with 40 µCi/ml [3H]methylthymidine (25 Ci/mmol, Amersham) was added. The extracellular virus was collected after 48 h and purified as described previously (Mrdberg et al., 2001
). Purified virus was resuspended in PBS (137 mM NaCl, 2·7 mM KCl, 8·1 mM Na2HPO4 and 1·5 mM KH2PO4) containing 0·1% BSA and stored at -70 °C. The number of virus particles in the purified preparations was calculated based on the determination of DNA content (Karger et al., 1995
).
Binding of radiolabelled virus to cells.
Monolayers of L, gro2C, sog9 and sog9 EXT-1 cells in 96-well plates were pre-cooled for 30 min at 4 °C, washed twice with cold PBS and blocked for 1 h at 4 °C with PBSBSA. All virus strains were adjusted to contain the same number of virus particles per ml and then serially twofold diluted in PBSBSA. Aliquots of 50 µl of respective virus dilutions (200000 to 6000 particles per cell) were added in triplicate and the plates were left for virus adsorption for 5 h at 4 °C under continuous shaking. The cells were then washed three times in cold PBS to withdraw unadsorbed virions and lysed in 5% SDS. Radioactivity was determined by scintillation counting and the results were expressed as a percentage of the number of virus particles that was added to the cells originally.
Duplicate monolayers of L, gro2C and sog9 cells were either mock-or pre-treated with one or both of the enzymes heparinase I and chondroitinase ABC (Sigma) for 30 min at 37 °C, cooled for another 30 min at 4 °C and washed twice with cold PBS. The cells were blocked as described above and radiolabelled virus was added at the same multiplicity of attachment, 2·5x104 virus particles per cell, and adsorbed for 1 h at 4 °C. The rest of the procedure was carried out as described above.
Purification of HSV-1 gC.
The protein was purified from strain KOS321 as described previously (Mrdberg et al., 2001
).
Isolation of HS and CS chains and binding of gC.
To radiolabel HS and CS, subconfluent monolayers (50%) of cells were grown for 48 h (L cells) or 65 h (gro2C and sog9 EXT-1 cells) in the presence of 50 µCi/ml Na235SO4 (1325 Ci/mmol, NEN) in sulfate-free EMEM supplemented with antibiotics and 10% FBS. The cell-associated HS chains from L and sog9 EXT-1 cells and the CS chains from gro2C cells were isolated as described previously (Lyon et al., 1994 ). Isolation of CS from gro2C cells and HS from sog9 EXT-1 cells as well as the isolation of a mixture of both HS and CS from L cells was performed without using any GAG-degrading enzymes.
The binding assay of GAG chains to purified gC was performed as follows. Purified gC was serially fivefold diluted in PBS supplemented with 0·05% BSA (see Fig. 2 for concentration range used). Each dilution was mixed with approximately 4000 c.p.m. of 35S-labelled HS from L cells, 3000 c.p.m. of HS from sog9 EXT-1 cells, 1600 c.p.m. of CS chains or 2000 c.p.m. of a GAG mixture from L cells. The rest of the procedure was carried out as described earlier (Trybala et al., 1998 ).
To test the specificity of binding, CS was degraded by chondroitinase ABC. Briefly, 20 µl of gro2C-specific CS, which contained approximately 15000 c.p.m., was mixed with 5 U chondroitinase ABC and 25 µl PBS supplemented with 0·1% BSA. The mixture was incubated at 37 °C for 3 h and the enzyme was heat-inactivated at 80 °C for 5 min. The binding assay was performed as described above. The effect of increased NaCl concentrations on binding of gC to HS and CS was tested in a similar manner (Trybala et al., 2000 ), except that the concentrations of NaCl were adjusted to the desired molarities by adding a 2 M solution of NaCl in PBS buffer and that either 2 µg gC was tested for binding to HS and 5 µg gC was tested for interaction with CS.
Inhibition of gC binding to cell surface with heparin and desulfated heparin.
This assay was performed as described previously (Svennerholm et al., 1991 ; Trybala et al., 1994
) using the monoclonal antibody B1C1 for gC detection. In brief, purified gC was pre-mixed with increasing concentrations of heparin or selectively desulfated heparin fragments (a gift from D. Spillmann, Uppsala, Sweden). Then, the heparingC mixtures were left to adsorb to monolayers of L, gro2C and sog9 EXT-1 cells for 1 h at 4 °C, washed three times and the amount of bound gC was detected by ELISA after fixation of the monolayers with 0·25% glutaraldehyde.
Assessment of infectivity in GAG-deficient cells.
The HSV gC mutant strains, as well as KOS321 and gC-39, were titrated on GMK-AH1 cells and approximately 300 p.f.u. per strain was added in duplicate to monolayers of GMK-AH1, L, gro2C, sog9 and sog9 EXT-1 cells. The monolayers had been washed in DMEM medium before the virus was added. Virions were allowed to adsorb for 2 h at 37 °C. The cells were then washed three times in DMEM and 1% methylcellulose solution was added. The cells were incubated for 4 days and stained with crystal violet. The number of virus plaques was expressed as a percentage of plaques formed on GMK-AH1 cells.
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Results |
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Purified gC binds CS isolated from gro2C cells
Results shown in Fig. 1 suggested that gC mediated HSV-1 attachment to cell surface CS on gro2C cells. However, these data do not provide any evidence that gC alone can form a stable complex with CS, since other cellular or viral proteins might have contributed to the viruscell interactions. Therefore, a direct binding assay of purified gC and CS was performed. CS isolated from gro2C cells, or HS isolated from L or sog9 EXT-1 cells, was mixed with immunoaffinity purified gC and the GAGgC complexes formed were trapped on nitrocellulose filters. As shown in Fig. 2
, purified gC bound to CS as well as to HS. To ensure the specificity of the CSgC interaction, a subfraction of the CS preparation was pre-treated with chondroitinase ABC before mixing it with gC. The results (also shown in Fig. 2
) showed that this treatment completely abolished the ability of CS to interact with gC. However, gC bound higher amounts of HS from L cells and similar amounts of the same molecule from sog9 EXT-1 cells as compared to CS from gro2C cells. This finding suggests that, even in cells from the same origin, quantitative and/or qualitative differences between HS chains may exist and influence the gCHS interaction. Alternatively, the differences could be explained by the varying efficiency of GAG chain-labelling due to differences in GAG biosynthesis between the two cell lines.
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Binding of gC to cell surface CS is inhibited by selectively desulfated heparins
The finding that gC bound CS in solution prompted attempts to evaluate binding of the protein to the surface of CS-expressing gro2C cells. Purified gC bound to all three cell types expressing surface GAG molecules. It was demonstrated earlier that binding of the gC+ virus to gro2C cells was more sensitive to inhibition by HS as compared to L cells (Banfield et al., 1995b ). To further characterize differences between gCHS and gCCS interactions, we inhibited gC binding to the cell surfaces of L, gro2C and sog9 EXT-1 cells by heparin as well as selectively desulfated heparin (Fig. 3
). Binding of gC to CS-expressing gro2C cells was more sensitive (approximately 10 times) to inhibition by heparin as well as by the selectively desulfated heparin, as compared to the HS-exposing L and sog9 EXT-1 cells (Fig. 3
, compare b to a and c). In addition, N-desulfated heparin was equally efficient as an inhibitor as heparin on gro2C cells as well as on sog9 EXT-1 cells, a finding that implies that N-linked sulfates on this molecule contribute little to gC binding (Fig. 3
, b
and c
). Although the lack of N-sulfate contribution to inhibition of CS binding of gC is explained by the absence of such sulfates on the CS chains, the experiments demonstrate a similarity in this property between the two cell lines expressing either HS or CS. No difference in inhibition by 2-O- and 6-O-desulfated heparin in gro2C cells was found.
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An intact N-terminal part of gC is important for efficient virus infection of gro2C cells
To determine whether gC was also required for efficient infection of CS-expressing cells, the gC+ (KOS321) and gC- (gC-39) virus strains were tested for their infectivity in L, gro2C, sog9 and sog9 EXT-1 cells (Fig. 5). The panel of gC mutant strains, used in the above attachment assays, was also included to characterize further the role of particular amino acid residues in gC for infection of CS-expressing gro2C cells compared to HS-expressing L and sog9 EXT-1 cells (Fig. 6
). To ensure equal amounts of infecting virus strains, all viruses were titrated on GMK-AH1 cells prior to assay, with the rationale that gC does not play a major part in attachment or infection of this cell line (Trybala et al., 1994
).
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Some mutants, such as I142T and K(114,117)A were slightly more impaired in infectivity on gro2C cells than on L and sog9 EXT-1 cells, suggesting a greater importance of some positively charged as well as hydrophobic amino residues for infectivity in this cell line. However, such differences might be revealed more easily in gro2C cells where infectivity was more gC-dependent. In conclusion, gC facilitated infectivity in cells expressing either CS or HS and the functional HSBD of gC was responsible for this facilitation also in gro2C cells, most likely due to an ability of the domain to accommodate CS features expressed in gro2C cells.
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Discussion |
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CS, a family of GAG molecules expressed ubiquitously in a plethora of human tissues (Bode-Lesniewska et al., 1996 ; Sorrell et al., 1999
; Zimmermann et al., 1994
), plays a significant role for several biological functions in vivo. In the nervous system, CS is involved in regulation of cell migration and axonal outgrowth (Bovolenta & Fernaud-Espinosa, 2000
). Furthermore, CS is a major component of the human skin in, for example, the basal membrane layer of the epidermis (Bode-Lesniewska et al., 1996
; Sorrell et al., 1999
). The major subtypes of CS (A, B and C) all bear similarities to HS in two ways: the repeating disaccharide units consist of amino sugars and hexuronic acids and these units show a variable degree of sulfation along the GAG chains. Due to the vast complexity of synthesis and modification of HS as well as CS chains and methodological limitations in sequencing sugar chains, specific domains functioning as initial receptors for viruses and other microbes have hitherto been difficult to delineate.
To define a role for gC during virus entry, previous attempts have utilized successfully the apical route of infection of polarized MDCK cells (Sears et al., 1991 ). This cell line was demonstrated recently to expose CS preferably on the apical surface in contrast to the basolateral areas, which mainly expose HS (Kolset et al., 1999
). Of the cells utilized in the present study, the HS-deficient cell lines gro2C and sog9 cells were sequentially isolated from mouse fibroblast L cells by selecting cell clones that were resistant to HSV-1 infection (Banfield et al., 1995a
; Gruenheid et al., 1993
). Here enzymatic removal of GAG molecules confirmed earlier results demonstrating the necessity of such molecules for virus entry in both L and CS-exposing gro2C cells and that infectivity of sog9 cells was unaffected by this treatment (Banfield et al., 1995b
; Laquerre et al., 1998
; Tal-Singer et al., 1995
). It is interesting to note that heparinase treatment alone did not profoundly affect virus attachment to HS/CS-expressing L cells, but that dual treatment, including chondroitinase, strongly reduced virus binding. Furthermore, in parallel with what was demonstrated earlier on polarized cells (Sears et al., 1991
), HSV-1 was dependent on a functional gC for attachment to as well as infection of the CS-expressing gro2C cells. It should be noted that in a previous study, using another gC- virus construct in a related experimental setting, no difference between the gC+ and the gC- viruses in attachment to gro2C cells was found (Banfield et al., 1995b
). Further studies comparing the gC- strains and rescued variants thereof might be needed to resolve the question.
HSV-1 infectivity in sog9 cells was found earlier to be rescued by transfection of these cells with the EXT-1 gene, an HS polymerase (Lind et al., 1998 ; McCormick et al., 1998
). This gave us the opportunity to compare a cell line expressing CS (gro2C cells) with cells expressing HS but no other GAG molecules (sog9 EXT-1 cells). In the attachment assay, binding of the gC+ viruses (KOS321 and gC rescue) were grossly similar in the two cell lines, suggesting that CS was at least as competent a molecule for initial binding of gC as was HS. This finding was supported by the comparable results from binding of gC to isolated CS and HS from these two cell lines. Furthermore, as judged by the results of a library of gC mutants, overlapping, but not identical, domains of gC were utilized for attachment to the CS- and HS-expressing cells, respectively. For both HS and CS interactions, a functional domain of gC consisting of positively charged arginine residues interspersed with hydrophobic amino acids was essential. In addition to the importance of I142 for HS binding reported previously (M
rdberg et al., 2001
), the T
F substitution at position 146 was found to affect virus attachment and infectivity severely in both gro2C and sog9 EXT-1 cells. Thus, hydrophobic interactions seem, in addition to electrostatic forces, to be decisive for CS as well as HS binding to gC.
When interactions between purified gC and GAG molecules and inhibitions thereof were studied, differences between CS and HS binding to gC were found. Our data evaluating the effect of increasing NaCl concentrations on gC complexes formed with HS or CS, where the gCCS complex was more sensitive to an increasing NaCl concentration than the gCHS complex, suggested a difference in the contribution of electrostatic forces between gCHS and gCCS interactions. This was in line with earlier data (Banfield et al., 1995b ), indicating that HSV-1 virions bound more weakly to gro2C cells compared to the HS-expressing L cells. Furthermore, inhibition by heparin and desulfated derivatives showed a much stronger effect on binding of purified gC to the CS-expressing cells as compared to HS-expressing cells. These results suggest that gC displays different binding affinities versus HS and CS, a feature demonstrated earlier for another GAG-binding protein, HCII (Tollefsen, 1994
).
The ability of gC to interact with both HS and CS could be understood if HS and CS were shown to share some structural features, such as similar gC-binding motifs. A prerequisite for such a comparison would be the definition of gC-binding sequences of HS and CS, respectively. In a previous work, we have shown that the gCHS interaction was sulfate-dependent and that a minimal requirement was a heparin dodecamer containing at least one 2-O- and one 6-O-sulfate, possibly localized to the same disaccharide unit (Feyzi et al., 1997 ). A similar dependence on disulfated disaccharides might also be important for proteinCS interactions, as suggested by a recent report on platelet factor 4 interactions with this family of GAG molecules (Petersen et al., 1999
). Although the CS molecules present on gro2C cells remain to be characterized, our preliminary results by removal of cell surface CS by specific chondroitinases suggested that structural features, which are removed by chondroitinase AC, but not B or C, were essential for infectivity of the gC+ KOS321 strain (unpublished observation). However, development of sequencing methods of CS chains, as has been achieved for HS, will be necessary to answer the question of whether HS and CS expose similar gC-binding motifs.
When analysing results of virus attachment as well as infectivity on L cells exposing both HS and CS chains, one striking feature was noticed: the gC-dependency that was obvious on the two cell lines exposing either of the GAG molecules was no longer discernible. Although we cannot explain this phenomenon at the present level of understanding, we suggest that the complexity introduced by the presence of dual receptors (HS and CS) with different gC affinities offers a greater possibility for other HS-binding viral proteins, such as gB, to display functional redundancy. Furthermore, the fact that expression and composition of CS and HS molecules in cell models utilized for studies of gC function in attachment and infectivity was, hitherto, given little attention might be of relevance for the contradictory results presented using similar experimental systems (Griffiths et al., 1998 ; Sears et al., 1991
).
The significance of the gCCS interaction demonstrated here for in vivo infection remains to be elucidated, as is also the case for gCHS binding. However, the finding of a direct dependency on gC for HSV attachment to as well as infection of a CS-expressing cell line is intriguing in light of recent findings of gC requirement for replication in human skin implants in a mouse model (Moffat et al., 1998 ) and demonstration of CS expression in such tissues (Bode-Lesniewska et al., 1996
; Sorrell et al., 1999
; Zimmermann et al., 1994
).
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Acknowledgments |
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References |
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Received 21 August 2001;
accepted 16 October 2001.