2 Department of Virology, University of Göteborg, Guldhedsgatan 10B; S-413 46 Göteborg, Sweden; and 3 Faculty of Health Sciences, School of Dentistry, Nørre Alle 20 DK-2200 N, Denmark
Received on September 22, 2003; revised on February 2, 2004; accepted on March 5, 2004
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Abstract |
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Key words: attachment / heparan sulfate / O-linked / plaque size
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Introduction |
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Being a highly glycosylated protein, gC-1 contains nine N-linked glycosylation sites of which at least eight are utilized and equipped with complex type glycans exclusively, ranging from di- to tetraantennary structures (Olofsson et al., 1999; Rux et al., 1996
). In addition, gC-1 contains numerous O-linked glycans, most of which are localized in two pronase-resistant clusters in the N-terminal part of gC-1 (Biller et al., 2000
; Dall'Olio et al., 1985
; Olofsson, 1992
). One possible function of these O-linked glycans is to contribute to an extended shape of gC-1, thereby facilitating contacts between virion-associated gC-1 and its different ligands (Stannard et al., 1987
). The clustered O-linked glycans of gC-1 may also have other functions, including direct glycoepitope signaling to effectors of the immune system. In this context it is intriguing that another herpes virus, for instance, bovine herpes virus type 4, encodes a virus-specific glycosyltransferase, ß-1,6-N-acetylglucosaminyltransferase (core 2 transferase), which is engaged in formation of immunologically active selectin receptors of O-linked glycan nature (Markine-Goriaynoff et al., 2003
; Vanderplasschen et al., 2000
). Finally, the O-glycosylated stretches of gC-1 in the vicinity of, say, the heparan sulfatebinding domain (Mårdberg et al., 2001
) may affect its binding specificity.
The biosynthesis of O-linked glycans is initiated by the addition of N-acetylgalactosamine (GalNAc) moieties to serine and threonine residues of a completely translated and folded polypeptide chain, a reaction catalyzed by 1 of at least 13 distinct cellular UDP-GalNAc:polypeptide GalNAc-transferases, which are localized throughout the Golgi apparatus (Hassan et al., 2000a). These GalNAc-transferase isoforms have different but partly overlapping peptide acceptor substrate specificities, and they are differentially expressed in cells and tissues (Gruenheid et al., 1993
), and gC-1 appears to serve as substrate for multiple human GalNAc-transferases (Biller et al., 2000
). This suggests that gC-1 will become O-glycosylated in any cell type included in the broad host cell range of HSV-1.
Here we report that a minor modification of a glycosylation signal of gC-1, that is, substituting alanines for two basic amino acids, resulted in major changes in the extent of O-linked glycosylation. Moreover, this modification changed the biological properties of mutant virus in two different ways, for instance, altered pattern of adsorption to primary glycosaminoglycan receptors and altered plaque morphology, indicating differences in the complex process of viral cell-to-cell spread.
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Results |
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This was further evaluated by comparing the electrophoretic mobilities of gC-1Rescue and gC-1(114K,117R)A produced in C1300 cells, a mouse neuroblastoma cell line with a general deficiency in galactosyltransferases (Lundström et al., 1987b). Pertinent here is that all O-linked glycans of gC-1 produced in C1300 cells constitute GalNAc monosaccharides, a few of which are sialylated, which should be compared with a variety of differently sized O-linked glycans up to at least tetrasaccharides that are associated with gC-1 produced in African green monkey kidney (GMK) cells (Lundström et al., 1987a
,b
). There was no demonstrable difference in electrophoretical mobility between gC-1Rescue and gC-1(114K,117R)A produced in C1300 cells (Figure 2), confirming that the difference in electrophoretic mobility indeed was caused by a higher content of O-linked glycans in gC-1(114K,117R)A: In C1300 cells, the possible extra O-linked glycosylation of gC-1(114K,117R)A was obviously insufficient to induce a corresponding shift for the gC-1 variants produced in C1300 cells. In addition, this result suggested that the difference in electrophoretic mobility between gC-1Rescue and gC-1(114K,117R)A, produced in GMK cells, reflected not only a larger number of but also physically larger O-linked glycans of mutant gC-1.
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Prediction of O-glycosylation sites in synthetic peptides by in vitro analysis of the O-glycosylation capacity of multiple human GalNAc-transferase isoforms
Synthetic peptides representing the O-linked glycosylation signal delimited by amino acids 106 and 124 were analyzed as substrates for individual, purified human GalNAc-transferases (Bennett et al., 1998; Biller et al., 2000
). A wild-type peptide (representing rescue virus) as well as a peptide containing the gC-1(114K,117R)A mutations were analyzed with GalNAc-T1, -T2, -T3, or -T6, respectively. The wild-type and the mutant peptide were readily glycosylated by GalNAc-T2, however, the reaction velocity with the mutant peptide was considerably higher than with the wild-type peptide (Table I). The three other enzyme isoforms investigated demonstrated only low catalytic activities with both the wild-type and mutant gC-1 peptide substrates (data not shown).
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We also investigated whether the mutations induced affected the plaque morphology of infected GMK, L cells, and Gro2C cells (Figure 7), because this is a phenomenon largely related to late functions of HSV-1 glycoproteins in the infectious cycle (reviewed by Johnson and Huber, 2002; Rajcani and Vojvodova, 1998
). We found that the plaques generated by HSV-1gC-1(114K,117R)A were almost 50% smaller than those formed by HSV-1Rescue (p < 0.001). The difference in plaque size between HSV-1gC-1(114K,117R)A and HSV-1Rescue was observed for GMK and L cells as well as Gro2C cells. This similarity between the three cell types was in contrast to the finding that the different attachment rate between mutant and wild-type virus was evident for only one of the investigated cells, Gro2C. This supports the notion that the changed plaque morphology on one hand and attachment rate to Gro2C on the other hand each are caused by different and independent consequences of the altered O-linked glycosylation signal. It is important that although the parental gC-1 null virus strain, designated gC-39, produced syncytial plaques (syncytial and nonsyncytial morphology depicted in Figure 8), we found that neither the plaques induced by HSV-1gC-1(114K,117R)A nor by HSV-1Rescue were syncytial, suggesting that no alterations in the membrane fusion capacity of HSV-1 glycoprotein were involved in the plaque size transition between these two latter virus strains. As an extra control, we analyzed confluent plates with more than 200 plaques each of HSV-1gC-1(114K,117R)A and HSV-1Rescue using a gC-specific monoclonal antibody. Interestingly, we found only a few nonstained gC-1-negative revertant plaques from plates infected with mutant as well as rescue virus, but as expected these plaques were of the same syncytial, large phenotype as the parent gC-1 negative virus strain. Altogether, the results indicated that the O-glycosylation peptide signal also affected viral spread in cell culture even in the absence of cell surface heparan sulfate.
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Discussion |
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The in vitro studies of O-linked glycosylation with individual GalNAc-transferases, using synthetic peptides as substrates, revealed some of the mechanistic details behind the increased O-linked glycosylation of gC(114K,117R)A. We choose GalNAc-T1, -T2, -T3, and -T6 because these are enzymes that function with naked peptide substrates and do not require prior GalNAc-glycosylation by other isoforms. They also represent the best characterized isoforms both in terms of specificity and expression patterns. GalNAc-T1 and -T2 further represent the most universally expressed isoforms (for a review, see Hassan et al., 2000a). Of the investigated GalNAc-transferases, only GalNAc-T2 demonstrated efficient activity with the wild-type and mutant peptide substrates, but the mutant peptide sequence was a significantly better substrate than the wild type. Furthermore, amino acid sequencing and MALDI-TOF MS showed that the mutant peptide was glycosylated with three moles of GalNAc (T111, S113, and T119), whereas the wild-type peptide was only glycosylated with two moles of GalNAc (T119 and S113). Thus the presence of positively charged amino acids among serine and threonine residues in the wild-type gC-1 peptide is likely to direct a lower density of O-glycosylation in this region, although the net difference was only one additional GalNAc residue attached with the mutant peptide.
It is clear that the total number of extra monosaccharides of O-linked glycans of gC-1(114K,117R)A compared with wild-type glycoprotein is considerably higher than what could be harbored in one extra O-linked glycan of gC-1(114K,117R)A. Considering that most of the O-linked glycans of gC-1, produced in GMK cells, constitute structures up to tetra- or possibly pentasaccharides (Lundström et al., 1987a), it appears unlikely that the contribution one or possibly two new O-linked glycans of gC-1(114K,117R)A, as suggested from the peptide model study, is sufficiently large to accommodate that many monosaccharides. Hence our results suggest that the induced mutations also affected the O-linked glycans of regions outside the peptide range (residues 106 and 124), most likely the domain delimited by amino acids 40 and 100, where numerous clustered O-linked glycans are harbored (Biller et al., 2000
; Olofsson, 1992
). This notion is in several ways in accordance with our current view of the temporal regulation of O-linked glycosylation. First, some GalNAc-transferase isoforms are not able to add GalNAc to a serine or threonine unless an adjacent serine or threonine residue is preglycosylated (Bennett et al., 1998
). Thus it is possible that the facilitated addition of the first few GalNAc residues in the mutated gC-1 may pave the way for subsequently acting GalNAc-transferases, resulting in further increase in O-glycan density of gC-1 outside the peptide substrate sequence studied in this report. GalNAc-transferases further contain lectin domains with specificity for GalNAc-glycopeptides (Hassan et al., 2000b
), and these are involved in enhancing the O-glycosylation density and possibly have other roles in directing O-glycosylation. Thus a seemingly marginal improvement of the O-glycosylation signal could have profound effects on the total content of O-linked glycans in a glycoprotein, such as gC-1. The notion that gC-1(114K,117R)A produced in cells with an intact O-glycosylation machinery contains larger O-linked glycans reflects a general increase in the size of each O-linked glycan rather than an increased number of O-linked glycans is supported by the comparison by the studies of gC-1Rescue and gC-1(114K,117R)A, produced in C1300 cells, containing only O-linked monosaccharides and to some extent sialylated GalNAc (STn) (Lundström et al., 1987b
).
The induced modification of the O-glycosylation changed the biological properties of HSV-1 in two ways. First, the decrease in the ability of HSV-1 to bind to the surface of chondroitin sulfateexpressing cells, which represents the initial step in a series of interactions between HSV-1 and different cellular receptors (Campadelli-Fiume et al., 2000; Mårdberg et al., 2002
). Second, the reduced size of the mutant virus plaques, which represents altered viral cell-to-cell spread, a more complex process as outlined in detail later. The chondroitin sulfate and heparan sulfatebinding sites of gC-1 are largely overlapping, and several arginine residues and a few hydrophobic amino acid residues in the peptide stretch aa 129160 are important determinants for binding to both types of glycosaminoglycan (Mårdberg et al., 2001
, 2002
; Trybala et al., 1994
). Thus the increased content of O-linked glycans in gC-1(114K,117R)A may interfere either sterically or electrostatically with the interactions between gC-1 and chondroitin sulfate. Therefore one function of K114 and R117 could be to maintain an optimum net charge of a domain of significance for chondroitin sulfate binding, achieved by negative modulation of O-glycan sialylation, further supported by their cationic nature. Still, it is possible that identical domains of gC-1 are involved in chondroitin and heparan sulfate binding; the interference by O-linked glycans may affect the access for each type of glycosaminoglycan differently. Hence the dynamic responsiveness of this O-glycosylation signal may be a viral means to moderate the level of gCchondroitin sulfate interactions.
Regarding the other biological effects of the modified O-linked glycosylation sequence, there is so far no specific role for gC-1 defined in the processes determining plaque size and viral cell-to-cell spread. The interplay between different HSV-1 gene products in promoting cell-to-cell spread is complex, involving several HSV-1 glycoproteins. Thus gD-1 seems to be a key factor for cell-to-cell spread of wild type HSV-1 strains in cultured cells (Cocchi et al., 2000), whereas cell-to-cell spread in keratinocytes may be mediated by gI/gE without any involvement of gD-1(Huber et al., 2001
; Johnson and Huber, 2002
). Moreover, syncytial mutants of HSV-1 may spread among cells owing to the activity of gK-1, possibly supported by gC-1 (Pertel and Spear, 1996
). If the function of gC-1 is regulatory, it may be difficult to assign a role to gC-1 in this multitude of interactions by use of deletion mutants. In spite of this, the present data suggest that the O-linked glycosylation signal of gC-1 constitutes one of several factors affecting the plaque size of HSV-1, probably by one or more of at least three conceivable ways. First, gC-1 interaction with cell surface chondroitin but not heparan sulfate is essential for the large plaque size phenotype of HSV-1. This explanation seems unlikely, because of the small decrease in chondroitin sulfate binding as observed for HSV-1gC-1(114K,117R)A compared with wild-type virus (Figure 4). Second, the O-linked glycans of gC-1 may modulate the cell-to-cell spread-promoting of other HSV-1 glycoproteins, including, gE/gI, gK, or gD. Third, the O-linked glycosylated peptide signal with its O-glycan array may itself be engaged in cell-to-cell spread or cell morphology activities affecting plaque size. This latter hypothesis is in line with both findings that (1) the degree of Ebola virusinduced cell detachment is proportional to the length of a used O-linked glycosylation signal in the Ebola virus glycoprotein (Simmons et al., 2002
), and (2) the finding that such signals are engaged also on nonviral cell detachment mechanisms (Chervenak and Illsley, 2000
).
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Materials and methods |
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The mutated HSV strain HSV-1gC-1(114K,117R)A has two amino residue substitutions, specifically lysine and arginine residues at positions 114 and 117 replaced with alanine residues. The HSV-1 virus designated HSV-1Rescue was used as a wild-type control, and this virus expresses an unmodified gC gene introduced into the HSV-1 gC null strain gC-39 (Holland et al., 1984). HSV-1gC-1(114K,117R)A and HSV-1Rescue were originally constructed for studies on the heparin sulfatebinding site of gC-1, and both viruses have been characterized in detail previously (Mårdberg et al., 2001
). To avoid possible differences in other genes than gC-1, the wild-type and the mutated gC-1 genes were inserted into gC-39 using identical protocols for homologous recombination to produce infectious virus clones. Three plaques each of mutant and wild-type virus were selected for further experimentation and the entire gene for gC-1 was control sequenced (Mårdberg et al., 2001
). The gC-1-null strain gC-39 produced essentially syncytial plaques, whereas the plaques of HSV-1gC-1(114K,117R)A and HSV-1Rescue were nonsyncytial. The number of physical viral particles was calculated as previously described (Mårdberg et al., 2001
). The virus particle/PFU ratio varied between 5001000, with no significant difference between mutant and rescue virus. The term gC(K114, R117)A is used for designation of the mutant gC-1 protein.
In vitro assessment of O-glycosylation capacity
Polypeptide GalNAc-transferase assays were carried out essentially as described using soluble, secreted, purified recombinant human GalNAc-T1, -T2, -T3, and -T6, expressed in insect cells (Bennett et al., 1996; Wandall et al., 1997
). Acceptor peptides, representing rescue gC-1, 106PKNNTTPAKSGRPTKPPGP124, and gC(K114, R117)A, 106PKNNTTPAASGAPTKPPGP124, were synthesized by Chiron Mimotopes (Victoria, Australia), and purified by the manufacturer to 99% purity. Briefly, the assays were performed in 25 µl total reaction mixture containing 25 mM cacodylate (pH 7.4), 10 mM MnCl2, 0.25% Triton X-100, 200 µM UDP- [14C]-GalNAc (3,700 cpm/nmol) (Amersham Pharmacia Biotech AB, Uppsala, Sweden) or 2000 µM UDP-GalNAc, 15 µg acceptor peptide for assays with GalNAc-T1, -T2 and -T3 and 0.78 to 25 µg acceptor peptide for assays with GalNAc-T6, and 0.25 mU of each purified GalNAc-transferase, respectively. Soluble, secreted forms of the human GalNAc-transferases analyzed were expressed in High Five cells, grown in cell-free media, and purified to near homogeneity with specific activities of 0.6 U/mg for GalNAc-T1, 0.5 U/mg for GalNAc-T2, 0.5 U/mg for GalNAc-T3, and 2.35 U/mg for GalNAc-T6 measured using peptides derived from MUC2, MUC1, and MUC7 human mucin tandem repeats as described previously (Wandall et al., 1997
). Products were quantified by liquid scintillation counting after chromatography on Dowex-1, octadecyl silica cartridges (Mallinkrodt Baker Inc, Philipsburgh, NJ), or HPLC (PC3.2/3 or mRPC C2/C18 SC2.1/10 Pharmacia, Smart System; Amersham Pharmacia).
Evaluation of GalNAc incorporation into peptides was performed by MALDI-TOF mass spectrometry (Voyager DE; AME Bioscience, Toroed, Norway) as described previously (Schwientek et al., 2002). Sites of incorporation was analysed by Edman degradation on an Applied Biosystems Procise HT 494 sequencer using glass fiber filters precycled with BioBrene Plus (Applied Biosystems, Foster City, CA) as immobilizing support. (N-terminal sequence analysis was carried out at the Protein Analysis Center, Kardinska Institute, Stockholm, Sweden.) The peptide samples were aliquots of HPLC fractions isolated in a mixture of acetonitrile, 0.1% trifluoroacetic acid and water. The samples were spotted onto the BioBrene Plus treated and precycled glass fiber filters. The PTH derivatives were separated on-line using a Brownlee Spheri-5PTH, 5 micron, 220 x 2.1 mm C18 column at 0.325 ml/min. N-terminal sequence analysis was carried out using a Procise HT instrument (Applied Biosystems) after application of samples in solution to precycled Biobrene-treated glass fiber filters (Applied Biosystems).
Metabolic labeling and immunoprecipitation of HSV-1 glycoproteins
Confluent layers of GMK cells in 6-well culture plates were washed two times with serum-free EMEM and infected with HSV-1Rescue or HSV-1gC-1(114K,117R)A at a m.o.i. of 3. The virus was allowed to adsorb to the cells for 1 h at 37°C, followed by addition of EMEM, containing antibiotics. After incubation in CO2-incubator for 4 hours 50 µCi/ml D-[6-3H]-glucosamine hydrochloride ([3H]-GlcN; Amersham Pharmacia, 30 Ci/mmol) was added. The cells were radiolabelled for 20 hours until harvest. The labeled culture supernatant was removed from cells and supplemented with 1% (v/v) NP40 and 1 mM AEBSF (4-(2-aminoethyl)-benzenesulfonyl, HCL:p-aminoethylbenzenesulfonyl fluoride, HCl; Calbiochem/Novabiochem, San Diego CA). Cells were detached using rubber policemen, washed twice with TBS (tris-buffered saline; 150 mM NaCl, 50 mM tris HCL, pH 7.5) and resuspended in 1 ml TBS containing, 1 % (v/v) NP40 and 1 mM AEBSF. Cell suspensions were sonicated on ice followed by centrifugation to remove cell debris.
Radioimmunoprecipitation was carried out essentially as previously described (Lundström et al., 1987a). Heat-inactivated formalin-fixed S. aureus, coated with anti-mouse antibodies (Dakopatts, Glostrup, Denmark), were mixed with anti-HSV-1gC-specific mouse monoclonal antibodies B1C1 or C4H11B for 2 hours at 4°C (Olofsson et al., 1983
; Sjöblom et al., 1992
). The antibody-staphylococcus complex was then washed three times in TTB [(TBS, containing 1% Triton X-100 and 0.1% bovine serum albumin (BSA)] and mixed with labeled culture supernatants or labeled cell lysates overnight at 4°C. The samples were washed twice in TTB and twice in TBS prior to analysis by homogenous SDS-PAGE (9.25% polyacrylamide gels) or gradient gel SDS-PAGE (NuPAGE 4-12%; Invitrogen; Carlsbad, CA) and fluorography (Amersham Pharmacia). In some experiments gC-1 was detected by immunoblot, using a monoclonal antibody specific for a linear epitope (Sjöblom et al. 1992
).
In some experiments radiolabeled gC-1 was affinity-purified as previously described and subjected to sialidase (V. cholerae, Behringwerke, Marburg, Germany; 100 U/ml) treatment at pH 4.5 for 2 h at 37°C. The glycoprotein and the released sialic acid were separated by gel filtration on short disposable Sephadex G-25 columns (Amersham Pharmacia).
N-Glycosidase (N-glycanase) digestion of immunoprecipitated glycoproteins
The enzymatic elimination of N-linked glycans was performed essentially as previously described (Olofsson et al., 1999). Briefly, [3H]-GlcN-labeled culture supernatants were immunoprecipitated as described above but after final wash the samples were resuspended in 10 µl 20 mM sodium phosphate buffer pH 7.5, containing 10 mM EDTA, 0,005% sodium azide, 1% (v/v) ß-mercaptoethanol, 1% (w/v) sodium dodecyl sulphonate (SDS) and incubated for 20 minutes at 37°C. The glycoprotein was denatured and eluted from the Staphylococci by boiling for 5 minutes followed by centrifugation. The samples were then diluted 10 times in 20 mM sodium phosphate buffer pH 7.5 containing, 10 mM EDTA, 10 mM sodium azide, 1% (v/v) ß-mercaptoethanol, 2% (v/v) NP40 to avoid SDS denaturation of PnGase. PnGase (6 µl of 200IU/ml; Roche Applied Sciences, Stockholm) was added to samples prior to incubation at 37°C for 20 hours. After acetone precipitation samples were finally analyzed on SDS-PAGE and autoradiography (Amersham Pharmacia).
Purification of extracellular virus
The purification of radiolabeled HSV-1 rescue and mutant strains were performed as previously described (Mårdberg et al., 2001). In brief, roller bottle cultures of GMK-AH1 cells were infected with either virus strain. Following virus adsorption the cells were washed, and 45 ml fresh EMEM supplemented with 40 µC/ml of [methyl-3H]- thymidine (25 Ci per mmol, Amersham) was added. The cells were incubated for further 48 h, and thereafter the virus was pelleted from culture medium and purified using a tree-step discontinuous gradient of sucrose as described (Karger et al., 1995
). Unless otherwise stated, purified virus was resuspended in PBS containing 0.1% BSA and stored at 70°C. The numbers of virus particles in the purified preparations were calculated based on the determination of the DNA content (Karger et al., 1995
).
Binding of radiolabeled virus to cells
Monolayers of GMK AHl, Gro2C cells, sog9 EXT-1 cells and L cells in 96-well plates were precooled for 30 min at 4°C, then washed twice with cold PBS and blocked for 1 h at 4°C with PBS-BSA. Different virus strains, adjusted to contain the same initial number of viral particles, were serially diluted in PBS-BSA, as indicated in the figure legends. Equal 50-µl portions of each virus dilution were added in triplicate, and the plates were left for virus adsorption for 5 h at 4°C under continuous agitation. Subsequently, the cells were washed three times in cold PBS to eliminate unadsorbed virions and lysed in 5% SDS. The radioactivity was determined by scintillation counting, and the results were expressed as the percentage of attached virions relative the number of virus particles originally added to the cells.
Virus plaque size assay on glycosaminoglycan-deficient cells
Monolayers of GMK-AHl and L cells in 6 well plates were infected with about 200 pfu per strain of the mutated HSV, gC(114K,117R)A, as well as the rescue strain (HSV-1Rescue). The monolayers were washed in complete medium, DMEM high glucose (L cells) and EMEM (GMK AHl) supplemented with penicillin and streptomycin prior to virus addition. The virions were allowed to adsorb at 37°C for 2 h, and thereafter the cells were washed three times in complete medium, and 1% methylcellulose solution was added. The cells were incubated for 3 or 4 days, as indicated in figure legends, and nonconfluent plaques were detected by black plaque assay with the gC-1-specific MAB B1C1 (Nilheden et al., 1983; Olofsson et al., 1983
). The plaque size was determined essentially as described by Baigent et al. (2001)
. In brief, the plates were photographed together with a ruler, using a computer-assisted CCD camera, and the largest diameter of each plaque was determined at 3x magnification, using the ruler scale for standardization. A mean value was calculated for each strain and cell line, and the statistical significance of the measurements was determined by using Student t-test.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Bennett, E.P., Hassan, H., and Clausen, H. (1996) cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-T3. J. Biol. Chem., 271, 1700617012.
Bennett, E.P., Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollingsworth, M.A., Merkx, G., van Kessel, A.G., and others. (1998) Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat. J. Biol. Chem., 273, 3047230481.
Biller, M., Mårdberg, K., Hassan, H., Clausen, H., Bolmstedt, A., Bergström, T., and Olofsson, S. (2000) Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C. Effects on glycoprotein properties. Glycobiology, 10, 12591269.
Bolmstedt, A., Hemming, A., Flodby, P., Berntsson, P., Travis, B.L., Linn, J.P.C., Ledbetter, J., Tsu, T., Wigzell, H., Hu, S., and Olofsson, S. (1991) Effects of mutations in disulfide bonds and glycosylation sites on the processing, CD4-binding, and fusion activity of HIV glycoproteins. J. Gen. Virol., 72, 12691277.[Abstract]
Campadelli-Fiume, G., Cocchi, F., Menotti, L., and Lopez, M. (2000) The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells. Rev. Med. Virol., 10, 305319.[CrossRef][ISI][Medline]
Chervenak, J.L. and Illsley, N.P. (2000) Episialin acts as an antiadhesive factor in an in vitro model of human endometrial-blastocyst attachment. Biol. Reprod., 63, 294300.
Cocchi, F., Menotti, L., Dubreuil, P., Lopez, M., and Campadelli-Fiume, G. (2000) Cell-to-cell spread of wild-type herpes simplex virus type 1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB). J. Virol., 74, 39093917.
Dall'Olio, F., Malagolini, N., Speziali, V., Campadelli-Fiume, G., and Serafini-Cessi, F. (1985) Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type1. J. Virol., 56, 127134.[ISI][Medline]
Friedman, H.M., Glorioso, J.C., Cohen, G.H., Hastings, J.C., Harris, S.L., and Eisenberg, R.J. (1986) Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates. J. Virol., 60, 470475.[ISI][Medline]
Friedman, H.M., Wang, L.Y., Fishman, N.O., Lambris, J.D., Eisenberg, R.J., Cohen, G.H., and Lubinski, J. (1996) Immune evasion properties of herpes simplex virus type 1 glycoprotein gc. J. Virol., 70, 42534260.[Abstract]
Gerber, S.I., Belval, B.J., and Herold, B.C. (1995) Differences in the role of glycoprotein C of HSV-1 and HSV-2 in viral binding may contribute to serotype differences in cell tropism. Virology, 214, 2939.[CrossRef][ISI][Medline]
Gruenheid, S., Gatzke, L., Meadows, H., and Tufaro, F. (1993) Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans. J. Virol., 67, 93100.[Abstract]
Hassan, H., Bennett, E.P., Mandel, U., Hollingsworth, M.A., and Clausen, H. (2000a) O-glycan occupancy is directed by substrate specificities of polypeptide GalNAc-transferases. In Ernst, B., Hart, B.W., and others (Eds.), Carbohydrates in chemistry and biology. Wiley-VCH, New York, pp. 271292.
Hassan, H., Reis, C.A., Bennett, E.P., Mirgorodskaya, E., Roepstorff, P., Hollingsworth, M.A., Burchell, J., Taylor-Papadimitriou, J., and Clausen, H. (2000b) The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N- acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities. J. Biol. Chem., 275, 3819738205.
Herold, B.C. and Spear, P.G. (1994) Neomycin inhibits glycoprotein C (gC)-dependent binding of herpes simplex virus type 1 to cells and also inhibits postbinding events in entry. Virology, 203, 166171.[CrossRef][ISI][Medline]
Herold, B.C., WuDunn, D., Soltys, N., and Spear, P.G. (1991) Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J. Virol., 65, 10901098.[ISI][Medline]
Hidaka, Y., Sakuma, S., Kumano, Y., Minagawa, H., and Mori, R. (1990) Characterization of glycoprotein C-negative mutants of herpes simplex virus type 1 isolated from a patient with keratitis. Arch. Virol., 113, 195207.[ISI][Medline]
Holland, T.C., Homa, F.L., Marlin, S.D., Levine, M., and Glorioso, J. (1984) Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins. J. Virol., 52, 566574.[ISI][Medline]
Huber, M.T., Wisner, T.W., Hegde, N.R., Goldsmith, K.A., Rauch, D.A., Roller, R.J., Krummenacher, C., Eisenberg, R.J., Cohen, G.H., and Johnson, D.C. (2001) Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes. J. Virol., 75, 1030910318.
Johnson, D.C. and Huber, M.T. (2002) Directed egress of animal viruses promotes cell-to-cell spread. J. Virol., 76, 18.
Karger, A., Saalmuller, A., Tufaro, F., Banfield, B.W., and Mettenleiter, T.C. (1995) Cell surface proteoglycans are not essential for infection by pseudorabies virus. J. Virol., 69, 34823489.[Abstract]
Liljeqvist, J.A., Svennerholm, B., and Bergstrom, T. (1999) Typing of clinical herpes simplex virus type 1 and type 2 isolates with monoclonal antibodies. J. Clin. Microbiol., 37, 27172718.
Lundström, M., Jeansson, S., and Olofsson, S. (1987) Host cell-induced differences in the O-glycosylation of herpes simplex virus gC-1. II. Demonstration of cell-specific galactosyltransferase essential for formation of O-linked oligosaccharides. Virology, 161, 395402.[CrossRef][ISI][Medline]
Lundström, M., Olofsson, S., Jeansson, S., Lycke, E., Datema, R., and Månsson, J. (1987) Host cell induced differences in O-glycosylation of the herpes simplex virus gC-1. I. Structures of non-sialylated HPA- and PNA-binding cabohydrates. Virology, 161, 385394.[ISI][Medline]
Mårdberg, K., Trybala, E., Glorioso, J.C., and Bergstrom, T. (2001) Mutational analysis of the major heparan sulfate-binding domain of herpes simplex virus type 1 glycoprotein C. J. Gen. Virol., 82, 19411950.
Mårdberg, K.T., Tufaro, E. Bergström, T. (2002) Herpes simplex virus glycoprotein C is necessary for efficient infection of chondroitin silfate-expressing gro2C cells. J. Gen. Virol., 83, 291300.
Markine-Goriaynoff, N., Georgin, J.P., Goltz, M., Zimmermann, W., Broll, H., Wamwayi, H.M., Pastoret, P.P., Sharp, P.M., and Vanderplasschen, A. (2003) The core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the African buffalo. J. Virol., 77, 17841792.
McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L.E., Dyer, A.P., and Tufaro, F. (1998) The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat. Genet., 19, 158161.[CrossRef][ISI][Medline]
Nilheden, E., Jeansson, S., and Vahlne, A. (1983) Typing of herpes simplex virus by an enzyme-linked immunosorbent assay with monoclonal antibodies. J. Clin. Microbiol., 17, 677680.[ISI][Medline]
Olofsson, S. (1992) Carbohydrates in herpesvirus infections. Apmis, 100, 8495.[ISI]
Olofsson, S., Biller, M., Bolmstedt, A., Mårdberg, K., Leckner, J., Malmström, B.G., Trybala, E., and Bergström, T. (1999) The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC. Glycobiology, 9, 7381.
Olofsson, S. and Bolmstedt, A. (1998) Use of lectins for characterization of O-linked glycans of herpes simplex virus glycoproteins. In Rhodes, J.M.M. (Ed.), Methods in molecular medicine: lectin methods and protocols. Humana Press, Totowa, pp. 175192.
Olofsson, S., Sjöblom, I., Lundström, M., Jeansson, S., and Lycke, E. (1983) Glycoprotein C of herpes simplex virus: characterization of O-linked oligosaccharides. J. Gen. Virol., 64, 27352747.[Abstract]
Pertel, P.E. and Spear, P.G. (1996) Modified entry and syncytium formation by herpes simplex virus type 1 mutants selected for resistance to heparin inhibition. Virology, 226, 2233.[CrossRef][ISI][Medline]
Rajcani, J. and Vojvodova, A. (1998) The role of herpes simplex virus glycoproteins in the virus replication cycle. Acta Virol., 42, 103118.[ISI][Medline]
Rux, A.H., Moore, W.T., Lambris, J.D., Abrams, W.R., Peng, C., Friedman, H.M., Cohen, G.H., and Eisenberg, R.J. (1996) Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus. J. Virol., 70, 54555465.[Abstract]
Schwientek, T., Bennett, E.P., Flores, C., Thacker, J., Hollmann, M., Reis, C.A., Behrens, J., Mandel, U., Keck, B., Schafer, M.A., and others. (2002) Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila. J. Biol. Chem., 277, 2262322638.
Serafini-Cessi, F., Dall'Olio, F., Pereira, L., and Campadelli-Fiume, G. (1984) Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC. J. Virol., 51, 838844.[ISI][Medline]
Simmons, G., Wool-Lewis, R.J., Baribaud, F., Netter, R.C., and Bates, P. (2002) Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence. J. Virol., 76, 25182528.
Sjöblom, I., Sjögren-Jansson, E., Glorioso, J.C., and Olofsson, S. (1992) Antigenic strucure of the herpes simplex virus type 1 glycoprotein C: demonstration of a linear epitope, situated in an environment of highly conformation-dependent epitopes. Apmis, 100, 229236.[ISI][Medline]
Sommer, M. and Courtney, R.J. (1991) Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC. J. Virol., 65, 520525.[ISI][Medline]
Stannard, L.M., Fuller, A.O., and Spear, P.G. (1987) Herpes simplex virus glycoproteins associated with different motphological entities projecting from the virion envelope. J. Gen. Virol., 68, 715725.[Abstract]
Trybala, E., Bergström, T., Svennerholm, B., Jeansson, S., Glorioso, J.C., and Olofsson, S. (1994) Localization of a functional site of HSV-1 gC-1 involved in binding to cell surface heparan sulfate. J. Gen. Virol., 75, 743752.[Abstract]
Wandall, H.H., Hassan, H., Mirgorodskaya, E., Kristensen, A.K., Roepstorff, P., Bennett, E.P., Nielsen, P.A., Hollingsworth, M.A., Burchell, J., Taylor-Papadimitriou, J., and Clausen, H. (1997) Substrate specificities of three members of the human UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3. J. Biol. Chem., 272, 2350323514.
Vanderplasschen, A., Markine-Goriaynoff, N., Lomonte, P., Suzuki, M., Hiraoka, N., Yeh, J.C., Bureau, F., Willems, L., Thiry, E., Fukuda, M., and Pastoret, P.P. (2000) A multipotential beta-1,6-N-acetylglucosaminyl-transferase is encoded by bovine herpesvirus type 4. Proc. Natl Acad. Sci. USA, 97, 57565761.