Department of Virology, Göteborg University, Guldhedsgatan 10b, S-413 46 Göteborg, Sweden1
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA2
Author for correspondence: Tomas Bergström. Fax +46 31 827032. e-mail tomas.bergstrom{at}microbio.gu.se
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Abstract |
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Introduction |
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At least two HSV envelope glycoproteins, glycoprotein B (gB) and C (gC), may mediate virus binding to cell surface HS (Herold et al., 1991 , 1994
; Gerber et al., 1995
; Laquerre et al., 1998
). The finding that HSV-1 mutants deficient in the expression of both gB and gC (Herold et al., 1994
) as well as gC-null (gC-) constructs lacking a putative heparin-binding domain of gB (Laquerre et al., 1998
) were severely impaired in HS-dependent attachment indicated that other envelope glycoproteins are less important during the virus attachment phase. A dominant role for gC over gB during virus attachment was suggested by the ability of some gC- but not gB-reactive antibodies to block virus binding (Fuller & Spear, 1985
; Svennerholm et al., 1991
). Furthermore, gC- mutants showed, in contrast to gB- mutants, an impairment in binding to some cell cultures (Homa et al., 1986
; Herold et al., 1991
, 1994
; Tal-Singer et al., 1995
). Hence, gC could be regarded as the principal attachment protein, acting through its HS-binding ability, although gB provides functional redundancy that might be important during infection of some cell types such as neurons (Immergluck et al., 1998
).
HSV-1 gC contains, as do many heparin-binding proteins, several clusters of basic residues that enable an electrostatic interaction with the negatively charged sulfate and carboxylate groups on the HS molecule. In this report, we have characterized a major HS-binding domain by performing site-directed mutagenesis on all of the positively charged residues and some of the hydrophobic residues in the N-terminal domain of gC. Results from binding assays of purified gC with different mutations to isolated HS chains showed that a cluster of arginine residues surrounding the Cys127Cys144 disulphide bridge (Rux et al., 1996 ) were required for this interaction and that Ile142 substantially contributed to the binding. Furthermore, in cells that were enzymatically pre-treated in order to reduce the number of exposed HS chains, the same HSV-1 gC mutants which were impaired in HS-binding also showed reduced attachment and infectivity.
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Methods |
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DNA purification.
gC-39 DNA, used for co-transfection, was purified from extracellular virions grown in GMK-AH1 cells. Briefly, cells cultured in 1 litre roller bottles were infected with virus at an m.o.i. of 1 p.f.u. per cell and incubated at 37 °C for 2 h. Cells were then rinsed once with EMEM and incubated for a further 48 h in 40 ml of fresh EMEM. Medium was collected and clarified by centrifuging at 2000 r.p.m. for 10 min. Virus was pelleted from the supernatant by centrifugation at 140000 g for 1 h, resuspended in 2 ml of TrisEDTA (TE) buffer containing 10 mM TrisHCl and 1 mM EDTA, pH 7·5, and disrupted with a solution containing 0·5% SDS, 10 mM EDTA and 0·2 M NaCl. Viral DNA was then purified by phenolchloroform extraction and precipitated with ethanol. DNA from HSV-1 gC mutants, used for sequencing and Southern blot analysis, was obtained from extracellular virions grown in plastic bottle cultures of GMK-AH1 cells. Virus was pelleted by ultracentrifugation and the pellet was resuspended in 400 µl of TE buffer. DNA was then purified using the QIAamp Blood kit (Qiagen). All restriction enzyme digestions and ligations of plasmid DNA were performed following standard recombinant DNA techniques (Maniatis et al., 1982 ).
Construction and identification of HSV-1 gC mutants.
The pGC plasmid comprising the gC gene (Homa et al., 1986 ) was cleaved with BsaAI (at nucleotide 605) and NheI (at nucleotide -34). The 639 bp fragment encoding the major part of antigenic site II of gC was inserted into the polylinker segment of pALTER between the SmaI and XbaI restriction enzyme sites. The resulting plasmid, pAltgC, served as a matrix for further mutations. Site-directed mutagenesis was performed using the Altered Sites II in vitro Mutagenesis system (Promega). The template plasmid, pAltgC, was rendered single-stranded, according to the protocol of the Altered Sites system, using the helper phage R408. To enhance mutagenesis, the selection primer was used at a 1:10 and a 1:500 dilution for each mutant single-stranded polynucleotide. Mutagenesis primers were designed to convert two neighbouring positively charged amino acids (arginine or lysine) to alanine or to introduce a single amino acid mutation (Fig. 1
). For detection purposes, each primer also carried a new restriction enzyme site. Once mutagenesis was confirmed by enzyme analysis, pAltgC was digested with BglII and EcoNI (at nucleotides -18 and 525, respectively, of the gC gene). The resulting 543 bp BglIIEcoNI fragment was inserted into its wild-type position in pGC, i.e. between the BglII and EcoNI sites, and sequenced to determine the presence of the desired mutations and also the lack of any unwanted mutations. DNA sequencing was carried out using dye-terminator cycle sequencing and an ABI Prism 310 Genetic Analyser (Perkin Elmer).
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Reactivity of HSV-1 gC mutants with MAbs.
The reactivity of MAbs with HSV-1 gC mutant viruses was tested by an ELISA-based method, as described previously (Trybala et al., 1994 ). Briefly, duplicate monolayers of GMK-AH1 cells in 96-well plates were infected with the different virus constructs at an m.o.i. of 50 p.f.u. per cell. HSV-1 strain KOS321 was used as a positive control and gC-39 as a negative control. When CPE was complete, monolayers were fixed with 0·25% glutaraldehyde, and 100 µl EMEM containing either of the gC-specific MAbs was then added. Following incubation of the cells with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory) and, subsequently, substrate, the absorbance value of the supernatant was measured at 405 nm.
To determine whether the introduced mutations in gC affected the expression of gC in mutant virions, we used an ELISA in which purified virus particles from the different mutants, previously quantified by their DNA content, were used as antigens. In brief, 20 µg of each mutant virus, serially diluted in carbonate buffer (eight times in twofold steps), was added to 96-well plates in quadruplicate and left overnight at 4 °C. Thereafter, 100 µl PBS with 1% BSA containing either a gC-reactive MAb (C4H11B6), of which the affinity towards gC was unaltered by the mutations introduced in gC, or the gE-reactive MAb B1E6, was added in duplicate. Conjugate was added and the absorbance values were measured at 405 nm, as described above. Quantification of gC was performed by comparing the reactivity of MAb C4H11B6 at the different concentrations of virus constructs. In addition, a ratio of the virus concentrations giving identical absorbance values within linear parts of the curves for the anti-gC and anti-gE MAbs was calculated for each mutant virus.
Purification of HSV-1 gC.
GMK-AH1 cells in 1 litre roller bottles were infected with KOS321, gC-39 or HSV-1 gC mutants at an m.o.i. of 1 p.f.u. per cell and incubated at 37 °C for 36 h. The infected cells were centrifuged at low speed and distrupted by freezing. The extracellular virus was pelleted from the supernatant medium by centrifugation at 160000 g for 1 h. Virus and cell pellets were combined, resuspended in cold lysis buffer [0·02 M TrisHCl, 0·15 M NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, pH 7·5] and kept on ice for 1 h. The lysate was then clarified by centrifugation at 130000 g for 1 h. Since some gE contamination in our immunosorbent purified gC preparations had been noticed previously, lysates were first passed through an immunosorbent column containing the gE-specific MAb B1E6 (Bergström et al., 1992 ) and then run through a column with MAb C4H11B6 to adsorb gC. Subsequently the column was washed with 0·02 M TrisHCl buffer (pH 7·5) containing 0·5 M NaCl, 0·1% Nonidet P-40 and 1 mM EDTA and then again with the same buffer without detergent. Bound material was eluted with 0·1 M glycineHCl buffer (pH 2·4) and immediately neutralized with 1 M TrisHCl, pH 8·0. The fractions that contained the highest amounts of gC were pooled and centrifuged through 30 kDa filters (Palfiltron) to exchange the eluent buffer with PBS (137 mM NaCl, 2·7 mM KCl, 8·1 mM Na2HPO4 and 1·5 mM KH2PO4). The protein concentration in the purified gC preparations was determined by the standard Lowry method (BioRad). The differently mutated gC proteins were then subjected to electrophoresis in a preformed separation gel of 412% polyacrylamide (Novex) and stained with colloidal Coomassie blue.
Isolation of radiolabelled HS chains and binding of gC to HS.
To radiolabel glycosaminoglycans, subconfluent monolayers of HEp-2 and GMK-AH1 cells were grown for 48 h in the presence of Na235SO4 (50 µCi/ml, Amersham Life Sciences) in sulfate-free EMEM supplemented with antibiotics and 8% FBS for HEp-2 cells, and with 0·05% primaton RL substance and 2% newborn calf serum for GMK-AH1 cells. Cell-associated HS chains were isolated as described previously (Lyon et al., 1994 ). For the HS-binding assay, purified gC (0·5 µg) in 0·2 ml of PBS supplemented with 0·05% BSA was mixed with approximately 3000 c.p.m. of 35S-labelled HS chains isolated from HEp-2 and GMK-AH1 cells. The rest of the procedure was carried out as described previously (Trybala et al., 1998
).
Haemagglutinating activity (HA) of HSV-1 gC mutants.
HSV-1 can induce a gC-dependent, heparinase-sensitive HA of mouse erythrocytes and an assay to determine the HA of HSV-1 gC was carried out as described previously (Trybala et al., 1993 ). In brief, cell-associated (CA) and extracellular (EX) HA antigens were prepared from virus-infected GMK-AH1 cells and infectious medium, respectively. Erythrocytes were collected from C57BL/6 mice, approximately 2 months old, and washed with an isotonic solution of NaCl buffered with 0·02 M phosphates (Na2HPO4 and KH2PO4, pH 7·0). To determine HA, serial dilutions of EX and CA preparations were mixed with murine erythrocytes and results expressed as the highest virus dilution that induced haemagglutination.
Purification of radiolabelled EX virus.
GMK-AH1 cells in roller bottles were infected with mutant viruses, KOS321 or gC-39 at an m.o.i. of 3 p.f.u. per cell. After virus adsorption for 3 h at 37 °C, cells were washed twice with EMEM before the addition of 4550 ml of EMEM supplemented with [methyl-3H]thymidine (40 µCi/ml). Cells were incubated for 48 h and the culture medium was collected and centrifuged at 2500 r.p.m. for 25 min to remove cell debris. The supernatant was then centrifuged at 36000 g for 2 h and the virus pellet was covered with 0·2 ml of PBS and left at 4 °C overnight. The pellet was gently resuspended in PBS and purified on a three-step discontinuous gradient of sucrose, as described previously (Karger et al., 1995 ). Unless otherwise stated, purified virus was resuspended in PBS 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 the DNA content (Karger et al., 1995
). The expression of gC by mutated HSV-1 virions was confirmed by immunoblotting using purified virus particles as antigen and the polyclonal anti-gC rabbit serum KF922.
Binding of radiolabelled virus to cells pre-treated with heparinase.
The following experiment was performed to determine the degree of HS reduction (cleavage) on cell surfaces allowing optimal discrimination between gC-positive (KOS321) and gC-negative virus (gC-39) attachment. Duplicate monolayers of HEp-2 cells were pre-treated with serial twofold dilutions of heparinase ranging from 8 to 0·008 units/ml for 1 h at 37 °C. The cells were then cooled for 30 min at 4 °C, washed twice with cold PBS and blocked for 1 h at 4 °C with PBS supplemented with 1% BSA. The respective virus strains were added at identical titres (25000 virus particles per cell) and left for adsorption for 1 h at 4 °C with continuous shaking. Cells were then washed three times in cold PBS to remove unadsorbed virions and lysed in 5% SDS. Radioactivity was determined by scintillation and results were expressed as a percentage of the number of virus particles that attached to the mock-treated control cells. Binding of HSV-1 gC mutants to heparinase-treated cells was assayed in a similar manner except that duplicate monolayers of GMK-AH1 and HEp-2 cells were pre-treated with a selected concentration of heparinase (0·25 units/ml).
Infectivity of HSV-1 gC mutants on heparinase-treated cells.
Confluent monolayers of GMK-AH1 cells in 6-well plates were rinsed twice with 2 ml of EMEM and then treated for 1 h at 37 °C with 1 unit of heparinase resuspended in 1 ml EMEM containing 0·1% BSA. The cells were again rinsed and approximately 100 p.f.u. of the respective mutant virus in 1 ml of EMEM was added and left to adsorb for 30 min at 37 °C. Cells were then washed twice with EMEM and overlaid with 4 ml of 1% methylcellulose solution. After 3 days of incubation at 37 °C, cells were stained with crystal violet to visualize virus plaques. The results were expressed as the percentage of the number of virus plaques on enzyme-treated cells when compared to the number of virus plaques on mock-treated control cells.
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Results |
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A total of 13 gC mutants and a gC-rescued variant of gC-39 were constructed. Purified clones were subjected to Southern blot analysis to verify successful recombination (i.e. at the same genomic position as in the wild-type HSV-1 gC-positive strain) between the mutated gC gene and the gC-39 strain DNA. All mutant viruses were then sequenced (amino acids 11322 of the gC gene) to confirm the presence of the designed mutations. Sequence data for the virus mutants were as expected for all strains, except for mutant K(89,95)A+, which in addition to the designed mutations displayed an altered sequence between residues 75 and 80 (i.e. the sequence 75KTTPTE80 was unintentionally replaced with 75NPRAHL80), probably caused by the primer annealing to two separate but repetitive sequences in this region of the gC gene.
Expression of gC and the effect of mutations on the antigenic structure
gC, immunoprecipitated from all but two mutant strains, showed similar mobility in SDSPAGE as gC from the KOS321 strain, indicating that the introduced mutations had not caused any major alterations in the size of gC. Two gC mutants, K(89,95)A+ and N148A, showed slightly higher mobility due to the loss of one N-glycosylation site in each mutant (Olofsson et al., 1999 ). Similar patterns were observed when the gC proteins, purified from the respective virus mutant by immunoaffinity chromatography, were subjected to SDSPAGE (Fig. 2
). Coomassie blue staining of gC from mutants R(151,155)A and R(155,160)A was weaker because a lesser amount of protein was electrophoresed.
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gC preparations purified from mutants R(143,145)A and R(151,155)A were found to be profoundly impaired in binding HS derived from HEp-2 cells (Fig. 3). Their binding activities were comparable to those of the negative control. Furthermore, gC proteins derived from the R(129,130)A, I142A, R(145,147)A and R(155,160)A mutants were also shown to poorly interact with HS from both GMK-AH1 and HEp-2 cell types. Intermediate-to-efficient binders of HS were gC proteins mutated at residues Lys114 and Arg117, Arg117 and Lys120, and Arg135 and Arg139, while gC from the remaining mutants was unimpaired in HS-binding. Similar data were found with HS derived from GMK-AH1 cells (data not shown). These results indicated that the cationic and hydrophobic amino acid residues localized at the base of the Cys127Cys144 loop and close to the C-terminal region thereof were essential for HS binding.
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HSV-1 gC mutants deficient in HS binding showed reduced infectivity in heparinase-treated cells
To determine whether the observed impairment in attachment to cell surfaces with reduced amounts of HS might influence the infectivity of the different mutants studied, GMK-AH1 cells were treated with 1 unit/ml of heparinase before addition of virus. The results expressed as a percentage of the number of plaques observed in mock-treated cells are shown in Fig. 6. The HSV-1 gC-rescued mutant as well as the KOS321 strain showed almost no decrease in infectivity after pre-treatment of cells with this specific dose of heparinase. Mutated strains carrying substitutions for basic residues stretching from Lys75 to Lys120 as well as Arg135 and Arg139 exhibited little decrease in their infectivity. In contrast, the gC mutants R(129,130)A, I142T, R(143,145)A, R(145,147)A, R(151,155)A and R(155,160)A showed a profound decrease (more pronounced than that of gC-39) in their ability to infect the enzyme-treated cells. Hence, our interpretation is that virus mutants carrying gC defective in HS binding and attachment to cells were also impaired in their ability to infect GMK-AH1 cells exhibiting reduced amount of cell surface HS.
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Discussion |
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Although a detailed structural characterization of HSV-1 gC is lacking, the disulfide bonds between the cysteine residues of the protein have been resolved (Rux et al., 1996 ). The residues within antigenic site II involved in HS binding are situated at or near a 16 residue loop (Cys127Cys144), which is well-conserved among mammalian herpesviruses, as judged from reported DNA sequences (Fitzpatrick et al., 1989
). In PrV, the HS-binding domain of gC was shown previously to be composed of three functionally redundant units located in the vicinity of the homologous loop region of that protein (Flynn & Ryan, 1996
). The architecture of the HS-binding domain in HSV-1 gC shows a variation of a similar theme. In addition, other regions within the HSV-1 gC antigenic site II, such as Gly247 (Wu et al., 1990
), selected during preliminary mapping of the HS-binding site on gC by the use of mar mutants (Trybala et al., 1994
), may well participate in HS interaction, but this question was not addressed in the present work.
The HS-binding domain of gC, identified in the present work, has an amphipathic character and the hydrophobic residue Ile142, but not Val140, was found to be decisive for HS binding of the protein as well as important for virus attachment and infectivity. In addition, results from ongoing studies with a single mutant carrying a Phe146-to-Thr mutation suggest an importance of this hydrophobic residue (unpublished observation). Taken together, these findings indicate that the binding of gC to HS is not a purely electrostatic interaction and that nonionic forces may contribute to free energy of binding, as was reported previously to be the case for heparinbasic fibroblast growth factor interactions (Thompson et al., 1994 ).
The findings that gC binding to HS is difficult to saturate and that gB may replace gC function in HS-driven attachment to the cell surface (Herold et al., 1991 , 1994
; Gerber et al., 1995
; Laquerre et al., 1998
) pose a difficulty when analysing effects of minor structural alterations in gC on virus adsorption. Here, we screened restricted mutations of the gC gene in otherwise unaltered viruses in two attachment assays selected to enable a gB-independent evaluation of gC function. The results from the gC/HS-specific HSV-1 HA (Trybala et al., 1993
) showed that those mutants from which purified gC proteins were found to be defective in HS interaction were also impaired in binding to murine erythrocytes. Furthermore, low-dose heparinase treatment provided the means to study impairment of gC function during attachment to living cells, probably explained by fewer HS molecules being available for binding. Again, results were in agreement with those from the HS-binding abilities of gC proteins, with one exception: the R(135,139)A mutant, only slightly impaired in HS binding and HA activity, showed a more pronounced disability in attachment to heparinase-treated cells, suggesting that these residues, situated at the apex of the loop, may be important for an additional interaction during the attachment step. However, the only moderately reduced infectivity of this mutant suggests that the results should be interpreted with caution, considering the methodological differences that exist between these experimental assays.
In the complex early events of -herpesvirus infection, a dispensable role for gC for overall infection of cultured cells was shown for HSV, PrV and BHV-1 based on whole gene deletion mutants (reviewed by Spear, 1993
). In line with this, even the gC mutants that were almost devoid of HS binding, such as R(143,145)A, replicated well in cell cultures in the present study. However, when infectivity was assayed after low-dose heparinase treatment of cells, this mutant appeared to be even less infective than the gC- virus. This finding indicates that the gCHS interaction is also important for virus infectivity and that the presence of gC molecules impaired in HS binding might hinder the redundancy of gB, at least in a milieu where the number and/or length of HS chains is reduced. Under such conditions, a defect in the gCHS interaction might constitute a bottle-neck that reduces virus infectivity.
By using HS-specific antibodies, the in vivo expression of specific HS epitopes was shown to be limited and tissue-specific (van Kuppevelt et al., 1998 ). The ability of gC to fish out a subset of HS molecules (Feyzi et al., 1997
) suggested a specificity of this interaction to which both molecules contributed. The understanding of the biosynthesis of HS, of specific importance for HSV infectivity, was increased by the characterization of the EXT-1 gene, which is one of a family of HS-specific glycosyltransferases (Lind et al., 1998
; McCormick et al., 1998
). In addition, the report that later stages of HSV-1 entry mediated by gD binding to a family of immunoglobulin-like cell surface molecules (Cocchi et al., 1998
; Geraghty et al., 1998
) can be fully substituted by gD binding to HS chains modified by a specific isoform of 3-O-sulfotransferase (Shukla et al., 1999
) poses the question of a possible functional relationship between gD/gC and cell surface HS molecules. Such studies might benefit from the availability of virus mutants defective in HS interaction, but which carry only point mutations in gC.
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Acknowledgments |
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References |
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Received 5 February 2001;
accepted 18 April 2001.