Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Room 201, Memphis, TN 38163, USA1
Author for correspondence: Patrick Ryan. Fax +1 901 448 8462. e-mail pryan{at}utmem.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, the various gD receptors are viewed as coreceptors, because initial PRV attachment is mediated by the interaction of glycoprotein C (gC) with cell surface heparan sulfate (HS) chains that are linked to proteoglycan cores (HSPGs) (Flynn et al., 1993 ; Flynn & Ryan, 1996
; Mettenleiter et al., 1990
). Surprisingly, this step is nonessential for infection, since gC-null strains are viable and PRV can infect some cells that are unable to express HS (Karger et al., 1995
; Robbins et al., 1986
). Infection is very inefficient under these circumstances, however, and a major role of PRV gC is to localize the virus to the cell surface and thus promote gD binding to a nonHSPG coreceptor (Mettenleiter, 1989
; Spear et al., 2000
). Additionally, gC plays a role in PRV entry, because null mutants are also delayed in virus penetration (Mettenleiter, 1989
).
Our laboratory has shown previously that the HS-binding domain of PRV gC is composed of three discrete clusters of mostly basic amino acids that closely match proposed motifs of heparin-binding domains (HBDs) (Cardin & Weintraub, 1989 ; Flynn & Ryan, 1996
). Working with mutant viruses that retained only a single gC HBD, it was demonstrated that each HBD can independently mediate virus attachment that is resistant to PBS washes of the inoculated monolayers. A mutant lacking all three HBDs was removed from monolayers under the same conditions (Flynn & Ryan, 1996
). Thus, the HBDs appear to function redundantly, but Trybala et al. (1998)
showed that each differs in their ability to bind differentially sulfated derivatives of HS. Therefore, the different HBDs may bind to different HS ligands to mediate initial attachment of the various mutants. It has not been determined if different mutants bind to different proteoglycan cores.
There are two major families of membrane-bound HSPGs, the syndecans and the glypicans (reviewed by Bernfield et al., 1999 ). These HSPGs serve as coreceptors for a number of biologically important ligands and infectious agents (Bernfield et al., 1999
; Rostand & Esko, 1997
). Specificity of binding to HSPGs resides mostly in the sulfation patterns of the attached HS chains. The chains are generally 50150 disaccharide units in length and can be sulfated at several positions on the disaccharide units (Bernfield et al., 1999
; Lindahl et al., 1998
). All HS chains from a given cell type are believed to be similarly modified, regardless of the proteoglycan core to which they are attached; the modifications result in a somewhat periodic clustering of lowly sulfated regions interspersed among highly sulfated disaccharides (Lindahl et al., 1998
). It is a lack of distinguishing HS chains for the different HSPGs of a cell that may prevent any one from serving as a specific receptor and, hence, relegate it to a coreceptor status. As coreceptors, HSPGs are oligomerized through ligand binding and can subsequently concentrate or immobilize ligands on the cell surface or assist in signal transduction (Bernfield et al., 1999
).
Karger & Mettenleiter (1996) have shown previously that PRV can bind both integral and peripheral HSPGs, but did not identify any specific species due to the high molecular mass of intact HSPGs. Here, we have resolved some of the specific HSPGs that are bound by wild-type and mutant forms of gC. We have uncovered preferences of individual HBDs for specific HSPGs, some of which are probably members of the syndecan family, and have demonstrated the importance of a diverse repertoire of HBDs for the overall ability of gC to bind HSPGs. In addition, we have been able to correlate the HSPG-binding capacities of columns composed of a single mutant gC with the binding kinetics of the corresponding PRV mutant strains.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Virus strains expressing gC fused to the cellulose-binding domain (CBD) of exo-1,4--glucanase from Cellulomonas fimi were constructed as follows. To begin, the CBD-coding sequence in pET-38b(+) (Novagen) was modified by site-specific mutagenesis in two ways. First, five consensus sites for N-linked glycosylation addition were removed by replacing either threonine or serine in the sequence Asn-X-Ser/Thr with alanine. Second, a BamHI site was introduced at the 3' end of the CBD-coding sequence along with an amber stop codon. All alterations were confirmed by DNA sequencing. A 377 bp XhoIBamHI fragment encoding the modified CBD was then inserted into a plasmid containing the 3' portion of gC and downstream sequences, replacing a XhoIBamHI fragment corresponding to gC codons 421460; this plasmid was named p3'gCCBD. To generate full-length gCCBD fusions encoding wild-type or mutant HS-binding domains, the upstream coding sequence and 5' portion of gC alleles from PRV-Becker, PRV580, -581, -582 or -583 were inserted in-frame into p3'gCCBD. Each plasmid was then cotransfected along with genomic DNA from PRV509 into PK15 cells by the calciumphosphate method. As codons 2458 of PRV509 gC had been deleted, PRV509 does not, therefore, express gC (Flynn et al., 1993
). Furthermore, in screening for recombinants that restored gC expression, the extent of the gC deletion in PRV509 ensured that the full-length of each gCCBD fusion was crossed into the virus genome. Recombinant viruses were identified using goat anti-gC serum 282 (Ryan et al., 1987
) and horseradish peroxidase (HRP)-conjugated antibodies in the black plaque assay (Holland et al., 1983
). Black plaques were purified for each construct and one was chosen for further use. The resulting strains and their encoded fusion proteins were: PRV612, gCWTCBD; PRV613, gCHBD3CBD; PRV614, gCHBD2CBD; PRV615, gCHBD1CBD; and PRV616, gCHBD0CBD.
To confirm production of the correct gCCBD hybrid proteins, PK15 cells were infected with 10 p.f.u. per cell of the gCCBD strains and incubated from 6 to 16 h post-infection in DMEM supplemented with 2% foetal bovine serum and penicillin/streptomycin (DMEM/2%) with 50 µCi/ml (1·85 MBq/ml) [35S]methionine/cysteine (NEN Research Products). Clarified culture supernatants were prepared as described previously (Ryan et al., 1987 ) and the gCCBD proteins were immunoprecipitated with monoclonal antibodies M1, M7 or M16 (Hampl et al., 1984
). The immunoprecipitates were resolved by SDSPAGE and subjected to fluorography and autoradiography (Ryan et al., 1987
).
Production of gCCBD affinity columns.
For each gCCBD fusion strain, ten 100 mm dishes of confluent PK15 cell monolayers were inoculated with 20 p.f.u. per cell in DMEM/2% at 37 °C for 1 h. The inocula were then removed and monolayers were overlaid with 4 ml DMEM/2% and incubated at 37 °C. At 16 h post-infection, virus-free culture medium was prepared as described previously (Ryan et al., 1987 ) and applied to CBinD 300 cartridges (Novagen) that had been equilibrated with 5 column vols of cold 20 mM TrisHCl, pH 7·5. The columns were then washed with 5 vols of cold 20 mM TrisHCl (pH 7·5) and 800 mM NaCl.
After all experiments were completed, the amount of gCCBD protein bound to each cellulose column was determined by eluting the fusion proteins. Briefly, 1 ml ethylene glycol was added to each column and collected. A small portion of each eluate was added to sample buffer and the samples were resolved by SDSPAGE and transferred to nitrocellulose membranes, as described below. Western blots were probed with goat anti-gC serum 282 followed by the addition of HRP-conjugated anti-goat antibody. Proteins were detected by chemiluminescence; each column yielded an equivalent amount of protein (data not shown).
Extraction of HSPGs.
After 3 days of growth, confluent monolayers of PK15, MDBK or mouse L cells on 100 mm dishes were rinsed with cold PBS and extracted at 4 °C in 1 ml 10 mM TrisHCl, pH 7·4, 150 mM NaCl and 1% Triton X-100 supplemented with 1 mM PMSF. After rocking the dishes constantly for 1 h, the extraction buffer was collected and centrifuged at 10000 g for 1 h at 4 °C. The resulting supernatant was diluted with 3 ml DMEM/2% if it was to be applied to gCCBD columns. However, if it was to be digested directly with glycosaminoglycosidases and then used in SDSPAGE, it was left undiluted.
Application of cell extracts to gCCBD columns.
A 600 µl aliquot of the diluted cell extracts was applied to each gCCBD column and the flow-through fraction was collected and passed through each column twice more before being saved. Each column was rinsed with 5 vols of cold 20 mM TrisHCl (pH 7·5) and the first 500 µl was collected and added to the flow-through fraction because this portion of the rinse contained a significant amount of HSPGs (data not shown). Five vols of 20 mM TrisHCl, pH 7·5, 400 mM NaCl were then applied to the columns, but only the first 1 ml from each was collected as the eluate because nearly all of the HSPGs that eluted from the column were in this fraction (data not shown). In some experiments, elution was achieved by sequentially applying 5 vols of 20 mM TrisHCl (pH 7·5) containing 200, 250, 300, 350 or 400 mM NaCl. After each use, the columns were washed with 5 vols of cold 20 mM TrisHCl (pH 7·5) and 800 mM NaCl to prepare them for future experiments.
Heparitinase and chondroitinase digestion.
Samples were digested in 50 mM HEPES (pH 7·0), 1 mM CaCl2, 0·1% Triton X-100 and 1 mM PMSF. Heparitinase (0·5 mIU, Sigma) and chondroitinase ABC (2·5 mU, Sigma) were added and the samples were incubated at 37 °C for at least 2 h. After digestion, samples were precipitated with the addition of 40 µg tRNA (carrier) and 3 vols of ethanol. Precipitates were resuspended in SDSPAGE sample buffer.
SDSPAGE and Western blotting conditions.
Samples were resolved by 10% SDSPAGE and then transferred electrophoretically to nitrocellulose membranes in 25 mM Tris, 192 mM glycine and 20% methanol. The buffer for all membrane washes and incubations was 20 mM Tris, 150 mM NaCl and 0·05% Tween-20 (TBS-T). Membranes were blocked in TBS-T containing 5% nonfat milk for 30 min at room temperature before incubating with primary antibodies in TBS-T containing 0·25% gelatin. The following antibodies were used: mouse monoclonal antibody 3G10 (obtained from G. David, Leuven University, Leuven, Belgium, and from Seikagaku), 281-2 rat anti-syndecan-1, MSE-2 rabbit anti-syndecan-2 and MSE-3 rabbit anti-syndecan-3 (all provided by M. Bernfield, Harvard Medical School, Boston, MA, USA). After 1 h at room temperature or overnight at 4 °C, the membranes were washed with TBS-T and then incubated with HRP-conjugated secondary antibodies. Afterwards, membranes were washed and chemiluminescence (SuperSignal West Pico, Pierce) was used to detect the protein bands on X-ray film.
Attachment assay.
For attachment assays, duplicate PK15 cell monolayers for each time-point were infected with approximately 150 p.f.u. of virus in 1 ml DMEM/2%. After incubation at 37 °C for 15, 30, 45 or 60 min, one monolayer of each pair was washed three times with prewarmed (37 °C) DMEM/2% containing 5 µg/ml heparin, while the other monolayer was washed three times with prewarmed (37 °C) DMEM/2% alone. All pairs were overlaid with DMEM/2% containing 1% methylcellulose to promote plaque formation. About 36 h later, plaques were counted and the number of plaques on the heparin-washed monolayers was expressed as a percentage of the number of plaques on the monolayers washed with medium alone.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
To express the hybrid proteins for binding to cellulose columns, each gCCBD fusion was recombined into the virus genome at the gC locus. As a result, the hybrid proteins received appropriate N- and O-linked glycosylation and were harvested from the culture medium of infected cells. To verify the authenticity of the gCCBD hybrids, PK15 cells were infected with each hybrid strain and incubated with medium containing [35S]methionine/cysteine. The culture medium was then harvested, cleared of cells and virus and used in immunoprecipitations with a panel of monoclonal antibodies that recognize conformation-dependent epitopes of gC (Hampl et al., 1984 ). The samples were resolved by SDSPAGE and the resulting autoradiogram is shown in Fig. 3(C)
. The hybrid proteins were secreted in similar amounts and appeared to be properly modified as they migrated at about 97 kDa, slightly larger than the wild-type species (Ryan et al., 1987
). In all cases, the hybrid proteins were immunoprecipitated by each of the three monoclonal antibodies, indicating that the replacement of the gC transmembrane domain with the CBD had not drastically altered their conformations.
Individual HBDs of gC bind similar HSPGs, but to different extents
Equivalent amounts of the secreted hybrid proteins were bound to cellulose columns to create gC-affinity columns from wild-type and each of the mutant strains. To ascertain the binding capacities of the columns, equal amounts of PK15 or MDBK cell extracts were added to each column. The columns were washed with binding buffer and then eluted with 400 mM NaCl. Eluate and flow-through fractions were digested with heparitinase and chondroitinase ABC, resolved by SDSPAGE and analysed by Western blot analysis using antibody 3G10. The results are shown in Fig. 4; both blots were scanned and relative amounts in each lane were determined using Scion Image software (Scion). The gCWTCBD column bound greater than 90% of added HSPGs from either extract (Fig. 4A
). The gCHBD3CBD and gCHBD2CBD columns bound about 40% of added HSPGs and the gCHBD1CBD column bound substantially less, about 10%. The gCHBD0CBD column, with no functional HBDs, bound less than 5% of the HSPGs added and these could be eluted at a low salt concentration of 50 mM NaCl (data not shown). This was consistent with the HSPGs binding to the columns through their HS side-chains, since the gCHBD0CBD column exhibited little, if any, affinity. This was confirmed by first treating the extracts with heparitinase prior to their addition to a gCWTCBD column: all of the HSPGs shifted toward the flow-through fraction. However, if the extracts were first digested with chondroitinase ABC instead of heparitinase, the HSPGs retained their ability to bind to the column and appeared in the eluate (data not shown).
|
A mixture of three mutant gCs binds HSPGs better than any single mutant alone
The efficient binding of HSPGs by the gCWTCBD column may have been due to the presence of all three different HBDs. Accordingly, we constructed a new column, gCMIXCBD, in which equal amounts of gCHBD3CBD, gCHBD2CBD and gCHBD1CBD were used. Importantly, the column was composed of the same total amount of fusion protein as the other columns.
The same amount of PK15 cell extract that was used in the experiment depicted in Fig. 4(A) was applied to the gCMIXCBD column. Eluate and flow-through fractions were collected, digested, separated by SDSPAGE and blotted with 3G10 antibody as before (Fig. 5
). The gCMIXCBD column bound 67% of the total sample added, more than any of the mutant gCCBD columns. Therefore, an expanded HBD repertoire appeared to increase the efficiency of HSPG binding. Still, the efficiency of the gCMIXCBD column fell short of the gCWTCBD column efficiency. This may have been due to the presence, at least potentially, of three functional HBDs in every wild-type species compared to only one in each of the mutants.
|
|
Reduced binding ability for HSPGs can impair mutant virus entry of cells
PRV gC-null mutants are defective not only for initial virus attachment, but for virus penetration as well (Flynn et al., 1993 ; Mettenleiter, 1989
). This is due presumably, at least in part, to a reduced efficiency in locating and binding to a coreceptor through gD, a necessary step in virus entry. We measured the binding of wild-type or mutant virus to a non HSPG coreceptor by washing inoculated monolayers with medium containing 5 µg/ml heparin; by this process, virions bound only to HSPGs were removed. The heparin-washed monolayers were then compared to those washed only with culture medium. As shown in Fig. 7(A)
, 90% of input wild-type virions gained resistance to heparin challenge by as early as 15 min after inoculation. None of the mutant strains retaining a single gC HBD reached a coreceptor with wild-type kinetics, even though all three approached wild-type binding levels by 1 h post-infection. Strains PRV580 and -581 exhibited intermediate phenotypes, with 6570% of input virions resisting heparin challenge at each time-point over 45 min of inoculation. PRV582, which retains only the most amino-terminal HBD (i.e. HBD1) showed a significant delay in reaching a coreceptor, with about one-third of the input virions acquiring heparin-resistance within 15 min and less than 60% by 30 min. Therefore, the attachment kinetics of the mutant strains were similar to the HSPG-binding abilities observed with the corresponding gCCBD columns (Fig. 4
) and indicated that reduced affinity for HSPG receptors delayed virus binding to non HSPG coreceptors.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wild-type gC proficiently bound all of the HSPGs that we detected by Western blot analyses with antibody 3G10. However, HBD2 and -3 preferentially bound 60 K, but bound 45 K poorly and, to an even worse extent, 70 K from MDBK cells. These were somewhat surprising results as it is generally assumed that all HS chains of a given cell type are modified by the same cellular machinery to the same extent (Carey, 1997 ). Our results suggest that either this is not the case in MDBK cells or that gC binds some HSPGs through additional, core-specific features, which has been suggested for other HSPGligand interactions (Rapraeger & Ott, 1998
). Alternatively, topological determinants may influence the accessibility of the HS chains. Various heparin-binding proteins have been shown to bind specific HS sequences (Bernfield et al., 1999
); our findings support the idea that topological differences might also preferentially direct the ligand to a particular proteoglycan core (Carey, 1997
).
It is not uncommon for heparin-binding proteins to contain multiple HBDs, although it appears in some cases that one of the HBDs is responsible for the majority of binding activity (Barkalow & Schwarzbauer, 1991 ). This may explain the presence of three functional HBDs in the amino terminus of PRV gC. Even though our laboratory has shown previously that any one of the three HBDs can lead to virus attachment that is resistant to PBS washes of the inoculated monolayers (Flynn & Ryan, 1996
), results here suggest that HBD2 and -3 are the dominant domains, leaving HBD1 to serve an ancillary role in virus binding. The overall binding capacities of columns composed of gCHBD2CBD or gCHBD3CBD fusion proteins were equivalent and significantly higher than the binding capacity of a gCHBD1CBD column. However, the gCHBD2CBD and gCHBD3CBD columns were distinguished from one another by their NaCl elution profiles. While we described the elution of HSPGs at higher salt concentrations as indicative of higher binding affinity for the gCHBD3CBD column, this characterization may be somewhat misleading. As noted by Trybala et al. (2000)
, proteinHS bonds are more stable in high salt buffers as the relative contribution of nonionic interactions increases. ProteinHS bonds that are dependent mostly on ionic interactions are broken more readily at lower salt concentrations. Thus, the NaCl elution profiles of the gCHBD2CBD and gCHBD3CBD columns may indicate that HBD2 bound HS predominantly through ionic interactions, while HBD3 relied more on nonionic interactions. This is consistent with past analyses showing that PRV581 virions, which contain only HBD2 of gC, preferentially bound highly sulfated derivatives of heparin, while PRV580 virions used HBD3 only to bind undersulfated heparin derivatives with greater affinity (Trybala et al., 1998
). Because HS chains exhibit clusters of lowly sulfated stretches intermingled with highly sulfated saccharides (Lindahl et al., 1998
), the dual nature of the sulfation preferences of HBD2 and -3 may ensure that a suitable ligand is always nearby for gC-mediated attachment.
Results obtained with the gCMIXCBD column emphasized further the importance of a diverse repertoire of HBDs in mediating binding to HS. Overall, the salt elution profile of the mixed column was representative of the individual profiles and was shifted to elution at lower salt concentrations compared to the wild-type column. Still, some HSPGs eluted at the highest salt concentrations and we would suggest that these represented individual HSPGs bound simultaneously to multiple fusion proteins bearing different HBDs. If this is true, such cross-linking was apparently stronger than any cross-linking that occurred on a column composed uniformly of a single HBD. How intermolecular cross-linking would compare to any potential intramolecular cross-linking by the wild-type species cannot be determined clearly by our assays.
The major function of gC is to mediate the initial attachment of PRV to cells and our in vitro-binding assays correlated with the attachment phenotype of each gC mutant. If gCHSPG interactions only facilitated initial virus attachment, then the identification of specific HSPGs bound by gC might be inconsequential. However, gC-null mutants are also defective in virus entry and we have been able recently to separate the role of gC in virus penetration from its role in attachment (unpublished data). Thus, the identity of bound HSPGs is potentially important because different properties, such as cytoskeletal association and the mediation of signal transduction, have been attributed to the various syndecans (Bernfield et al., 1999 ; Rapraeger & Ott, 1998
). While we cannot point to a particular HSPG yet, it is possible that a property associated with a specific HSPG facilitates PRV penetration, if not attachment, through gC.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L. & Lose, E. J. (1992). Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annual Review of Cell Biology 8, 365-393.
Bernfield, M., Götte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. & Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annual Review of Biochemistry 68, 729-777.[Medline]
Cardin, A. D. & Weintraub, H. J. R. (1989). Molecular modeling of proteinglycosaminoglycan interactions. Arteriosclerosis 9, 21-32.[Abstract]
Carey, D. J. (1997). Syndecans: multifunctional cell-surface co-receptors. Biochemical Journal 327, 1-16.[Medline]
David, G., van der Schueren, B., Marynen, P., Cassiman, J.-J. & van den Berghe, H. (1992). Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells. Journal of Cell Biology 118, 961-969.[Abstract]
Flynn, S. J. & Ryan, P. (1996). The receptor-binding domain of pseudorabies virus glycoprotein gC is composed of multiple discrete units that are functionally redundant. Journal of Virology 70, 1355-1364.[Abstract]
Flynn, S. J., Burgett, B. L., Stein, D. S., Wilkinson, K. S. & Ryan, P. (1993). The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells. Journal of Virology 67, 2646-2654.[Abstract]
Hampl, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K.-O. & Kaplan, A. S. (1984). Characterization of the envelope proteins of pseudorabies virus. Journal of Virology 52, 583-590.[Medline]
Holland, T. C., Sandri-Goldin, R. M., Holland, L. E., Marlin, S. D., Levine, M. & Glorioso, J. C. (1983). Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay. Journal of Virology 46, 649-652.[Medline]
Karger, A. & Mettenleiter, T. C. (1996). Identification of cell surface molecules that interact with pseudorabies virus. Journal of Virology 70, 2138-2145.[Abstract]
Karger, A., Saalmuller, A., Tufaro, F., Banfield, B. W. & Mettenleiter, T. C. (1995). Cell surface proteoglycans are not essential for infection by pseudorabies virus. Journal of Virology 69, 3482-3489.[Abstract]
Kim, C. W., Goldberger, O. A., Gallo, R. L. & Bernfield, M. (1994). Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns. Molecular Biology of the Cell 5, 797-805.[Abstract]
Klupp, B. G., Nixdorf, R. & Mettenleiter, T. C. (2000). Pseudorabies virus glycoprotein M inhibits membrane fusion. Journal of Virology 74, 6760-6768.
Lindahl, U., Kusche-Gullberg, M. & Kjellén, L. (1998). Regulated diversity of heparan sulfate. Journal of Biological Chemistry 273, 24979-24982.
Lories, V., Cassiman, J.-J., van den Berghe, H. & David, G. (1989). Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts. Journal of Biological Chemistry 264, 7009-7016.
Mettenleiter, T. C. (1989). Glycoprotein gIII deletion mutants of pseudorabies virus are impaired in virus entry. Virology 171, 623-625.[Medline]
Mettenleiter, T. C., Zsak, L., Zuckermann, F., Sugg, N., Kern, H. & Ben-Porat, T. (1990). Interaction of glycoprotein gIII with a cellular heparinlike substance mediates adsorption of pseudorabies virus. Journal of Virology 64, 278-286.[Medline]
Rapraeger, A. C. & Ott, V. L. (1998). Molecular interactions of the syndecan core proteins. Current Opinion in Cell Biology 10, 620-628.[Medline]
Robbins, A. K., Whealy, M. E., Watson, R. J. & Enquist, L. W. (1986). Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture. Journal of Virology 59, 635-645.[Medline]
Roizman, B. (1990). Herpesviridae: a brief introduction. In Fields Virology , pp. 1787-1793. Edited by B. N. Fields & D. M. Knipe. New York:Raven Press.
Rostand, K. S. & Esko, J. D. (1997). Microbial adherence to and invasion through proteoglycans. Infection and Immunity 65, 1-8.
Ryan, J. P., Whealy, M. E., Robbins, A. K. & Enquist, L. W. (1987). Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites. Journal of Virology 61, 2962-2972.[Medline]
Saunders, S., Jalkanen, M., OFarrell, S. & Bernfield, M. (1989). Molecular cloning of syndecan, an integral membrane proteoglycan. Journal of Cell Biology 108, 1547-1556.[Abstract]
Spear, P. G., Eisenberg, R. J. & Cohen, G. H. (2000). Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275, 1-8.[Medline]
Trybala, E., Bergström, T., Spillmann, D., Svennerholm, B., Flynn, S. J. & Ryan, P. (1998). Interaction between pseudorabies virus and heparin/heparan sulfate. Journal of Biological Chemistry 273, 5047-5052.
Trybala, E., Liljeqvist, J.-., Svennerholm, B. & Bergström, T. (2000). Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate. Journal of Virology 74, 9106-9114.
Turner, A., Bruun, B., Minson, T. & Browne, H. (1998). Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. Journal of Virology 72, 873-875.
Received 8 August 2001;
accepted 16 October 2001.