Department of Pediatrics and Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1657, New York, NY 10029, USA1
Author for correspondence: Betsy Herold. Fax +1 212 426 4813. e-mail betsy.herold{at}mssm.edu
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Abstract |
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Introduction |
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However, important serotype differences in the relative contribution of gC towards virus attachment have emerged. For most strains examined, deletion of HSV-1 gC (designated gC-1- viruses) markedly reduces virus binding (Herold et al., 1991 ; Immergluck et al., 1998
; Laquerre et al., 1998
; Tal-Singer et al., 1995; Griffiths et al., 1998
). In contrast, deletion of gC-2 does not result in loss of specific binding activity (particles bound per cell) or specific infectivity (p.f.u. per particle) when compared with the parental wild-type virus, HSV-2(G) (Gerber et al., 1995). Moreover, while gC-1- mutants show a marked lag in the kinetics of virus penetration, the gC-2- virus does not. Together, these results indicate that, in contrast to other alphaherpesviruses, the gC homologue does not play a major role in mediating attachment for HSV-2. This challenges the assumption that homologous glycoproteins play identical roles for HSV-1 and -2 and underscores the need to independently study both serotypes.
The finding that the deletion of gC-2 does not result in any loss in specific binding activity or infectivity suggests that gB-2, the other heparin-binding glycoprotein, mediates HSV-2 attachment. To directly test this hypothesis, a gB-2 deletion virus (gB-2-) and a doubly deleted virus (gB-2-xgC-2-) were constructed and characterized with respect to binding, entry and cell-to-cell spread.
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Methods |
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Construction and isolation of gB-2- and gB-2-xgC-2- viruses.
VgB2 cells were co-transfected with HSV-2(G) or gC-2- viral DNA and DNA from an engineered plasmid, designated pBEH-GH-I, which contains the gB-2 gene (UL27) disrupted at the unique SnaB1 site by the hygromycin/enhanced green fluorescent protein (EGFP) fusion protein under the control of the immediate early promoter of human cytomegalovirus (HCMV) constructed from pHygGFP (Clontech). This dual function marker vector allows for drug selection with the ability to identify positive transfectants using GFP as a fluorescent reporter. Co-transfections were performed using the Effectene Transfection reagent (Qiagen). Recombinants were selected on VgB2 cells for ability to grow in the presence of 800 µg/ml hygromycin and expression of EGFP. Selected recombinants were plaque-purified three times and subsequently working stocks of two clones, designated HSV-2(G) gB-2- and gC-2-xgB-2-, respectively, were selected for further analysis.
To confirm that the gB-2- viruses contain the EGFPhygromycin cassette disrupting the gB-2 gene, viral DNA was purified, digested with HindIII and separated by agarose gel electrophoresis. The DNA fragments were transferred to nitrocellulose and hybridized to the NotI fragment of UL27, which differentiates intact gB-2 (17·7 kb) from disrupted gB-2 (10·8 kb) or to a probe containing the EGFPhygromycin cassette. The lack of gB and gC expression was confirmed by Western blot analysis using a monoclonal antiserum to gB or gC (1123 and 1125, Goodwin Institute). Blots were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse IgG and developed using the ECL kit (DuPont), as described previously (Qie et al., 1999 ).
Purification and quantification of virus.
Virions were purified from Vero or VgB2 cells on dextran gradients, as described previously (Herold et al., 1991 ). For gB-2- and gB-2-xgC-2- viruses, which produce non-infectious progeny on non-permissive cells, Vero cells were inoculated at an m.o.i. of 5 p.f.u. per cell for gB-2- and 1015 p.f.u. per cell for gB-2-xgC-2- virus and purified on dextran gradients 18 h post-infection (p.i.). The m.o.i. refers to the titre on complementing VgB2 cells of virus stocks produced on the complementing cell line. Titres of the purified virus were determined by plaque assays on Vero and VgB2 cells. The number of virus particles was determined by comparing the amounts of VP5 or gD by densitometric scanning with slight modification of methods described previously (Tal-Singer et al., 1995
; Qie et al., 1999
; Gerber et al., 1995). Dilutions of each virion preparation were solubilized and polypeptides were fractionated by SDSPAGE. Gels were Coomassie blue- or silver-stained and the relative purity of preparations compared. The proteins were transferred to PVDF (Perkin-Elmer) by Western transfer in 20 mM Tris, 150 mM glycine and 20% methanol and probed with monoclonal antibodies (mAbs) 1103 (anti-gD, Goodwin Institute) or 10-H44 (anti-VP5, Fitzgerald Industries International). Both the Coomassie blue-stained gels (VP5 band) and Western blots (gD) were scanned and analysed using the GELDOC 2000 system (Bio-Rad ) linked to an IBM PC and the relative number of virus particles determined. The purity of the virus preparations from host cell proteins was also examined by probing the Western blots with a mAb for cellular
-actin (AC-15, Sigma).
Virus binding assays.
Cells were grown in 6-well dishes, pre-cooled to 4 °C, blocked in 3% BSA for 30 min and then exposed to serial twofold dilutions of purified virus (representing relatively equivalent numbers of particles for each virus based on a Coomassie-stained gel scanned for VP5 and corresponding to a range of 0·011 p.f.u. per cell for G) for 5 h at 4 °C. In pilot experiments, we found that binding reaches equilibrium after
5 h at 4 °C. The unbound virus was removed by washing the wells three times with cold PBS. Cells were counted and harvested in 200 µl per well of buffer containing 20 mM Tris, pH 7·5, 50 mM NaCl, 0·5% NP-40, 0·05% DOC and supplemented with complete protease inhibitors (Roche). The soluble fraction was separated by centrifugation at 16000 g in a microcentrifuge for 10 min at 4 °C. Equal portions of the input and cell-bound virus (soluble fraction) were separated by SDSPAGE and quantified by comparing relative virus particle numbers by densitometric scanning after Western blotting with anti-gD mAb (1103) as detailed above. Mock-exposed cells were scanned and the background subtracted. The blots were also probed with the anti-
-actin mAb (AC-15) to compare relative amounts of cellular proteins. To assess the ability of heparin or chondroitin sulfate C to inhibit binding, studies were conducted in the presence of these soluble glycosaminoglycans (Sigma).
Immediate early gene expression.
Cells were infected with virus (m.o.i. of 0·110 p.f.u. per cell or equivalent particle numbers). At 4 h p.i., the cells were harvested in 200 µl per well of buffer containing 20 mM Tris, pH 7·5, 50 mM NaCl, 0·5% NP-40, 0·05% DOC and supplemented with complete protease inhibitors. The soluble fraction was separated by centrifugation at 16000 g. Proteins were separated by SDSPAGE and ICP27 detected by immunoblotting with mAb 1113. The blots were scanned and analysed using the GELDOC 2000 system.
Infectious centre assays.
Infectious centre assays were performed as described previously (Herold et al., 2000 ; Roller & Herold, 1997
). Briefly, Vero or VgB2 cells (donor cells) were exposed to different viruses at 37 °C to allow entry (m.o.i. of 10 p.f.u. per cell based on titre on complementing cell line). Cells were washed with a low pH citrate buffer to inactivate residual extracellular virus 12 h after infection. Then, 45 h after infection, the infected cells were detached with trypsinEDTA, counted and
100 cells plated onto duplicate monolayers of uninfected cells in the presence of medium containing pooled human IgG (Sigma). The pooled human IgG neutralizes infection by virus released into the medium.
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Results |
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Binding of gB-2- and gB-2-xgC-2- virus to cells
To determine the impact of deleting gB-2 on virus attachment, the binding of purified HSV-2(G), gC-2-, gB-2- or gB-2-xgC-2- viruses was compared. The gB-2- viruses were purified from both Vero and VgB2 cells as a control. Proteins from each purified virus preparation were separated by SDSPAGE and the relative amount of VP5 quantified by optical density scanning of the VP5 band following staining with Coomassie blue. Based on this initial quantification, serial twofold dilutions (representing equivalent numbers of particles) of each virus were allowed to bind Vero cells for 5 h at 4 °C. Unbound virus was removed by washing the monolayers three times with PBS (Gerber et al., 1995 ; Herold et al., 1991
). The input and bound viruses were then quantified in parallel by scanning the gD band on Western blots prepared with the serial dilutions of virus input and cell lysates of bound virus. Blots were also probed for
-actin to compare the amount of cell lysate loaded in each lane and to examine the purity of the virus preparations. This approach allows for comparison of binding activity in a more physiologic range (
0·011 p.f.u. per cell) and in the absence of radiolabelling.
Quantifying the virus input by scanning gels for VP5 or blots for gD yielded similar results for relative virus particle numbers (Fig. 2). The band intensities were in a linear range (r2=0·97 and 0·95, respectively, by linear regression). A densitometric scanning reading of 132±8 units for gD corresponds to an m.o.i. of 1 p.f.u. per cell for HSV-2(G). Representative blots showing input and bound virus for HSV-2(G) and gB-2- virus (purified on Vero cells) probed with anti-gD and anti-
-actin are shown in Fig. 3(a)
. No actin was detected in purified input virus but similar quantities were detected in the cell lysates. Blots for the other virus mutants probed with anti-gD are shown in Fig. 3(b)
. Three independent experiments for each virus were performed and the results are summarized in the accompanying graph (Fig. 3c
). As anticipated, deletion of gC-2 alone does not result in any loss in binding activity, confirming studies published previously (Gerber et al., 1995
). In contrast, deletion of gB-2 resulted in a marked reduction in virus binding activity. Complementation of the gB-2-deleted viruses by growth on VgB2 cells partially restored binding activity. Less than 5% of gB-xgC-2- virions bound to cells, although the input tested was lower because of difficulties in purifying as much double-negative virus. Using non-purified virus stocks, the gB-2-xgC-2- virus showed a greater reduction in specific binding activity compared with the gB-2- virus (data not shown).
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Discussion |
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HSV-2, therefore, appears to be unique among alphaherpesviruses with respect to the role of its glycoproteins in virus attachment. For most strains of HSV-1 and the related animal herpesviruses, pseudorabies virus (PrV) and bovine herpesvirus-1 (BHV-1), binding is mediated principally by the interactions of gC homologues with cell surface heparan sulfate (Griffiths et al., 1998 ; Herold et al., 1991
; Mettenleiter et al., 1990; Okazaki et al., 1991
). Whether HSV-2 evolved differently and whether this difference contributes to cell and tissue tropism remains to be determined. It should be noted, however, that gB homologues for several of the human beta- and gammaherpesviruses, such as HCMV, human herpesvirus (HHV)-7 and HHV-8, bind heparan sulfate (Akula et al., 2001
; Boyle & Compton, 1998
; Secchiero et al., 1997
).
HSV-1 viruses deleted of gC-1 are impaired in binding and kinetics of penetration but remain infectious, presumably because the heparan sulfate binding activity of gB-1 is sufficient to mediate virus binding (Herold et al., 1994 ). In contrast, in the absence of gC, the gB homologues for PrV and BHV-1 do not appear to mediate binding to heparan sulfate. PrV variants deleted in gC (PrV gC-) bind via a heparan sulfate-independent mechanism, suggesting that PrV gB does not productively interact with heparan sulfate. This is supported by the findings that PrV gC- strains are relatively heparin-resistant; only at concentrations of
100 µg/ml does soluble heparin inhibit PrV gC- infection of Vero cells (Mettenleiter et al., 1990
). Moreover, PrV gC- mutants infect wild-type or heparan sulfate-deficient mutant cells to a similar extent (Karger et al., 1995
). PrV gB binds to heparinSepharose only if gC is also present in the viral envelope, possibly due to glycoproteinglycoprotein interactions (Mettenleiter et al., 1990
). These results may reflect the lack of a heparin-binding domain within the sequence of PrV gB; analysis of the linear amino acid sequence of PrV gB supports this notion. HSV-1 and -2 gB proteins are only
50% homologous to PrV gB at the amino acid level (Mettenleiter & Spear, 1994
; Spear, 1993
). Another possible important difference is that PrV gB is proteolytically processed into subunits that remain linked via disulfide bonds, whereas HSV gB proteins are not (Mettenleiter & Spear, 1994
). Similarly, BHV-1 gB, which is processed in a manner similar to PrV gB, also fails to mediate heparan sulfate binding, although isolated BHV-1 gB exhibits heparin-binding activity (Klupp et al., 1997
).
Relatively high local concentrations of basic amino acids are characteristic of the heparin-binding domains of proteins. Analysis of the amino acid sequences at the N-termini of the HSV-1 and -2 forms of gB and gC reveals that there is a short lysine-rich region in gB and a longer lysine- and arginine-rich region in gC (Bzik et al., 1986 ; Dowbenko & Lasky, 1984
; Frink et al., 1983
; Stuve et al., 1987
; Swain et al., 1985
). Notably, the amino acid sequences of gB and gC are highly conserved between the two serotypes except at the N-termini and particularly in these basic regions. Interestingly, gC-1 differs considerably from gC-2 in this region, including an insertional/deletional variation (Spear, 1993
). If these basic regions mediate the binding to heparan sulfate, differences in sequences might contribute to differences in binding activity.
The observation that there are serotype differences in the relative contribution of gB and gC to virus binding is supported by known differences in epidemiology, cell tropism and susceptibility to inhibitors of virus binding. For example, HSV-1 is more likely to cause oral labial infections and sporadic encephalitis, whereas HSV-2 commonly causes genital lesions. This epidemiological observation is supported by in vitro studies that show that HSV-1 binds to human synaptosomes and glial cells more efficiently than does HSV-2, whereas HSV-2 binds cervical cells more efficiently than does HSV-1 (Vahlne et al., 1979 , 1980
). Neomycin, poly(L)-lysine and platelet factor 4 inhibit binding of HSV-1 but not HSV-2 (Campadelli-Fiume et al., 1990
; Herold & Spear, 1994
; Langeland et al., 1988
, 1990
; Oyan et al., 1993
). Conversely, selectively O-desulfated heparins preferentially inhibit binding of HSV-2 (Herold et al., 1996
). The observations that heparan sulfate is a common receptor for both serotypes but that the two preferentially bind different cell types and differ in susceptibility to select inhibitors of binding may be explained if gC-1 and gB-2 preferentially recognize distinct structural sequences of heparan sulfate differentially expressed by different cells. Thus, genital tract epithelial cells may express heparan sulfate sequences preferentially recognized by gB-2, whereas oral mucosal cells may express sequences preferentially recognized by gC-1.
In addition to playing the major role in mediating HSV-2 attachment, gB-2 is also essential for penetration and cell-to-cell spread. Deletion of gB-2 results in non-infectious virions that fail to enter, as evidenced by an inability to detect any immediate early gene expression even at a relative m.o.i. of 10 p.f.u. per cell, and fail to laterally spread, as evidenced by an inability to form infectious centres on non-complementing cells. These essential functions in virus-mediated fusion events are not unique to HSV-2 as all of the gB homologues studied to date have been shown to play similar roles (Spear, 1993 ). Whether or how the heparan sulfate-binding activity of gB-2 contributes to its role in mediating these fusion events cannot be determined from these studies. For HSV-1, it was shown that deletion of the heparin-binding lysine-rich region (residues 6876) resulted in viruses that, while infectious, exhibited impaired entry and cell-to-cell spread (Laquerre et al., 1998
). Recombinant viruses carrying mutations in the putative heparin-binding domain of gB-2 are under construction to determine the contribution of heparan sulfate interactions towards entry and cell-to-cell spread.
Together these studies demonstrate a major role for gB-2 in HSV-2 binding and an essential role in entry and cell-to-cell spread. Thus, gB-2, along with the other essential envelope glycoproteins, gD and gH-gL, are important targets for development of novel antiviral therapies, including topical microbicides that might prevent sexual or perinatal transmission of HSV-2 by blocking virus entry.
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Acknowledgments |
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References |
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Received 18 February 2002;
accepted 26 April 2002.