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Journal of Virology, July 2006, p. 6629-6636, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.01988-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

N- and 6-O-Sulfated Heparan Sulfates Mediate Internalization of Coxsackievirus B3 Variant PD into CHO-K1 Cells

Andreas E. Zautner, Birgit Jahn, Elke Hammerschmidt, Peter Wutzler, and Michaela Schmidtke*

Institute of Virology and Antiviral Therapy, Medical Centre of the Friedrich Schiller University Jena, Hans Knoell Str. 2, PF, D-07740 Jena, Germany

Received 19 September 2005/ Accepted 3 April 2006


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ABSTRACT
 
Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO3(6OSO3)]n, but not basic fibroblast growth factor binding to [HexUA-GlcNSO3-HexUA-GlcNSO3-IdUA(2OSO3)]n, was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO3(6OSO3)]n is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.


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INTRODUCTION
 
Group B coxsackieviruses (CVB) belonging to the family of nonenveloped picornaviruses utilize the coxsackievirus-adenovirus receptor (CAR) to bind to and enter into host cells (5). Small depressions surrounding the fivefold axis, the so-called canyons formed by the viral capsid proteins VP1, VP2, and VP3, bind the CAR (24). Receptor binding induces conformational changes which facilitate the internalization of viral RNA into host cells (18, 26). Additionally, human decay accelerating factor (hDAF/CD55) functions as an attachment but not an entry receptor for CVB1, -3, and -5 (6, 32). Recently, cell surface heparan sulfate proteoglycans (HSPG) were identified as additional receptors for the CVB3 variant PD (CVB PD) (38). Using HSPG for infection, CVB3 PD also replicates in CAR-lacking cell lines, e.g., CHO-K1, BHK-21, RD, and L929 (29).

HSPG consist of a polydisaccharide chain tethered to serine residues of defined core proteins by a linking tetrasaccharide composed of xylose-galactose-galactose-glucuronic acid (12). N-Acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) residues are added alternatingly to the linker tetrasaccharide. Several steps of modifications of GlcNAc and GlcA then follow, including N-deacetylation and N-sulfation of GlcNAc, C-5 epimerization of GlcA to L-idoronic acid (IdoA), 2-O-sulfation of the uronic acid, and 6-O- and 3-O-sulfation of D-glucosamine (GlcN) residues. The resulting high molecular diversity of heparan sulfate (HS) chains enables many specific interactions with very different proteins and glycoproteins, e.g., growth factors, cytokines, and human pathogens, including enveloped viruses (13, 22, 36). Cell surface HS were also shown to bind nonenveloped viruses, e.g., a variant of human rhinovirus 89 (HRV89) (37), echoviruses (15), swine vesicular disease virus (11), and Theiler's murine encephalomyelitis virus (25), belonging, like CVB3 PD, to the picornavirus family. The sulfated structural motifs of HS mediating binding or entry of picornaviruses are poorly known. Strong differences in viral replication in CHO cell mutants with different defects in heparan sulfate synthesis led to the assumption that CVB3 PD binds to specifically sulfated HS moieties.

During this study, the following tasks were performed: (i) the structural requirements, especially the sulfation pattern of HS necessary for CVB3 PD entry, were examined by using competition assays with growth factors binding to specifically sulfated HS sequences as well as with specifically desulfated heparins; (ii) the entry pathway of CVB3 PD while using HS was studied; and (iii) the kinetics of viral entry and the viral life cycle depending on the presence of CAR or HS as the receptor were investigated.


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MATERIALS AND METHODS
 
Cell lines and viruses. Chinese hamster ovary cells (CHO-K1; German Collection of Microorganism and Cell Cultures no. ACC-110) and heparan sulfate-negative, human CAR-transfected pgsD-677-hCAR cells (38) were grown in Dulbecco's modified Eagle's medium (Cambrex, Belgium) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. HeLa Ohio cells (Flow Labs) were propagated in Eagle's minimal essential medium supplemented with 10% neonatal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The test medium contained only 2% neonatal calf serum.

Stocks of CVB3 PD (35) and CVB3 H3 (20) were prepared in CHO-K1 and HeLa cells, respectively. Aliquots were stored at –80°C until use. Virus titers were determined on HeLa Ohio cells by end-point titration.

Compounds. Fully de-O-sulfated heparin, 2-O-desulfated heparin, 6-O-desulfated heparin, de-N-sulfated heparin (all from Neoparin Inc., San Leandro, Calif.), and heparin from bovine lungs (Sigma, Deisenhofen, Germany) were dissolved in sterile water (10 mg/ml) and stored at 4°C until dilution in test media. Recombinant human hepatocyte growth factor (rhHGF; R&D Systems, Wiesbaden, Germany) and basic fibroblast growth factor (bFGF; Sigma, Deisenhofen, Germany, and R&D Systems, Wiesbaden, Germany) were dissolved in Dulbecco's modified Eagle's medium (20 µg/ml) and stored at –20°C.

Stock solutions of ammonium chloride (pH 7.4; VEB Laborchemie, Apolda, Germany), chlorpromazine (Alexis, Grünberg, Germany), and sucrose (Leidholdt Biochemie, Kleinmachnow, Germany) were prepared in distilled water, stocks of monensin (Sigma, Deisenhofen, Germany) were prepared in ethanol, and stocks of bafilomycin A1 (Sigma, Deisenhofen, Germany) and nystatin (Serva Feinbiochemica, Heidelberg, Germany) were prepared in dimethyl sulfoxide, and all stock solutions were stored at 4°C until use.

Cytotoxicity assay and CPE inhibitory assay. The 50% cytotoxic concentrations of test compounds were determined on 2-day-old confluent CHO-K1 cell monolayers grown in 96-well microtiter plates 72 h after compound addition, as described previously (28). Cytopathic effect (CPE) inhibitory assays were carried out with CVB3 PD in CHO-K1 cells (38). Briefly, desulfated heparins as well as growth factors in test medium were added immediately before virus inoculation to confluent monolayers of CHO-K1 cells. Virus-induced CPE was scored spectrophotometrically after 2 days of incubation at 37°C.

To study the role of endosomal pH in CVB3 PD replication, CHO-K1 and pgsD-677-hCAR cells were preincubated with different concentrations of ammonium chloride (1.56 to 100 mM; dilution factor of 2) for 1 h at 37°C. Virus was added at a multiplicity of infection (MOI) of 10 50% tissue culture infective doses (TCID50)/cell, and incubation was continued for 3 h at 37°C and 5% CO2 together with ammonium chloride. Cells were then washed three times with test medium, and fresh medium was added. After incubation for a further 48 h at 37°C, virus-induced CPE was scored spectrophotometrically as described previously (28). Two experiments each, with six replicates per concentration, were performed.

Inhibition of viral antigen expression by inhibitors of endocytic pathways. The inhibitory activities of monensin (50 µM), bafilomycin (300 nM), sucrose (0.3 mM), chlorpromazine (10 µM), and nystatin (10 µg/ml) against CVB3 PD were comparatively studied in nearly confluent monolayers of HeLa, CHO-K1, and pgsD-677-hCAR cells grown on glass slides with 16 chambers. Heparin was used as a control compound. Inhibitors were added to the test medium and preincubated for 1 h at 37°C. Cells were infected at an MOI of 10 TCID50/cell, and incubation was continued for 3 and 4 h at 37°C for the HeLa and Chinese hamster ovary cell lines, respectively. The cells were then washed three times with test medium, and 100 µl of fresh medium without inhibitor was added and incubated for a further 4 h (HeLa cells), 20 h (pgsD-677-hCAR cells), or approximately 40 h (CHO-K1 cells). Afterwards, cells were fixed with a mixture of formalin-methanol and double-distilled water (10 ml/25 ml/65 ml) at room temperature for 20 min. CVB3 antigen-positive cells were detected with a CVB3-specific monoclonal antibody in an alkaline phosphatase-anti-alkaline phosphatase (APAAP) assay. Briefly, following three washings with phosphate-buffered saline, slides were incubated for 30 min with the CVB3-specific monoclonal antibody moAk 948 (Biermann Diagnostica, Germany). The cells were then washed three times with Tris-buffered saline containing 0.05% saponin and stained with an APAAP mouse detection kit (Dako, Germany) according to the manufacturer's instructions. If CVB3 antigen was present, a bright red color was observed in the cytoplasm of infected cells. Three separate experiments, each with two or three replicates per inhibitor, were performed.

Virus inactivation by exposure to low pH. Six samples each of CVB3 PD and CVB3 H3 in phosphate-buffered saline (200 µl) were adjusted to pH 4.5 by the addition of 0.5 M acetate buffer (pH 4), as described recently by Vlasak et al. (37). After 15 or 30 min of incubation at room temperature, three samples of each virus were neutralized with 0.5 M Na3PO4. Three untreated virus samples were used as controls and incubated for 30 min. Virus titers were determined by end-point dilution on HeLa cells.

Influence of time of virus removal on CVB3 PD antigen expression. To compare the time necessary to initiate CVB3 infection in cell lines with specific receptor expression patterns, nearly confluent monolayers of HeLa, CHO-K1, and pgsD-677-hCAR cells grown on glass slides with 16 chambers were inoculated with CVB3 PD at an MOI of 10. After 15 or 30 min or 1, 2, or 4 h of incubation at 4°C (allows only virus binding) or 37°C (permits virus binding and entry), the virus inoculum was aspirated from two chambers, cells were washed three times, and 100 µl of fresh test medium was added. Incubation at 37°C and 5% CO2 was then continued. Two chambers of CVB3 PD-infected cells were used as virus controls. To allow maximum virus attachment and entry, CVB3 PD was incubated in control chambers during the whole experimental time. One or two chambers of noninfected cells served as cell controls on each chamber slide. Seven (HeLa cells), 24 (pgsD-677-hCAR cells), or 48 (CHO-K1 cells) hours after virus inoculation, cells were fixed with a mixture of formalin-methanol and double-distilled water (10 ml/25 ml/65 ml) at room temperature for 20 min. Afterwards, APAAP staining was performed as described above to examine the influence of the time of virus removal on binding and/or entry by comparing the number of virus antigen-expressing cells after various times of virus removal with that for virus controls. Three independent experiments, each with two replicates per time point, were performed.

One-step growth experiments. HeLa, CHO-K1, and pgsD-677-hCAR cells were seeded in four-well plates. Three wells of each plate with 2-day-old cell monolayers were inoculated with CVB3 PD at an MOI of 10 TCID50/cell in 200 µl of test medium. One well of each plate was used as a cell control. After 1 h of incubation at 37°C and 5% CO2, the inoculum was aspirated from HeLa cells. The cell monolayers were washed three times, and 500 µl of fresh medium was added. Immediately thereafter (1 h) and 2, 3, 4, 5, 6, 7, 8, and 9 h after infection, one plate was frozen at –80°C for virus titration. Incubation of CVB3 PD-infected cells was continued at 37°C and 5% CO2.

For CHO-K1 and pgsD-677-hCAR cells, the same procedure was used, with some modifications. Both 1 and 2 hours after virus inoculation, one plate was washed three times to remove unbound virus and was frozen after the addition of 500 µl of test medium. Four hours after virus inoculation, the remaining plates were washed and supplemented with 500 µl of test medium. One plate was frozen immediately at –80°C and the others were frozen after 6, 24, and 48 h of incubation at 37°C and 5% CO2. Viral titers were determined by end-point titration on HeLa cells. To release the intracellular virus, infected cell monolayers were freeze-thawed three times before virus titer determinations were performed.


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RESULTS
 
Specific inhibition of CVB3 PD infection by growth factors binding to specifically sulfated HS motifs and by heparins with definite desulfation patterns. In our previous study, under-N-sulfated pgsE-606 cells (3) resisted CVB3 PD infection (38). Moreover, pgsF-17 cells were identified as the cell line with the minimum of HS modifications permissive for CVB3 PD infection. HSPG of pgsF-17 cells contain large amounts of N-sulfates and 6-O-sulfates but lack 2-O- and 3-O-sulfates (2). Therefore, we suggested that both N-sulfated and 6-O-sulfated HS are essential for interaction of HS with the virus capsid. To prove this hypothesis, competition assays with growth factors known as natural HS ligands as well as with chemically desulfated heparins were performed.

Control experiments showed that the inhibitors themselves were not toxic to cells at the maximum concentration used (Table 1).


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  		TABLE 1. Cytotoxicities and antiviral activities of polycations, polyanions, and growth factors against CVB3 PD in CHO-K1 cells

The competition of bFGF and rhHGF with CVB3 PD for binding to cell surface HS was compared. bFGF binds to HexUA moieties in proximity to N- but not 6-O-sulfated glycosamines within the oligosaccharide sequence [HexUA-GlcNSO3-HexUA-GlcNSO3-IdUA(2OSO3)]n, and rhHGF interacts with N- and also 6-O-sulfated glycosamines next to idoronic acid in its binding motif, [IdUA-GlcNSO3(6OSO3)]n (1, 2). Medium, bFGF, or rhHGF was added immediately before virus inoculation to CHO-K1 cells. After 48 h of CVB3 PD infection, cell viability rates in treated and mock-treated wells were determined. The results demonstrate (Fig. 1A) a dose-dependent inhibition of CVB3 PD replication by rhHGF, whereas the two tested bFGF preparations hindered the CVB3-induced CPE only weakly (Sigma, Germany) or not at all (R&D Systems, Germany) at 0.15 to 10 µg/ml.


Figure 1
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  		FIG. 1. Results from competition assays with HS-binding growth factors (A) and chemically modified heparins (B). Whereas rhHGF binding to the disaccharide [IdUA-GlcNSO3(6OSO3)]n markedly blocks virus infection, bFGF binding to a motif lacking 6-O-sulfate groups does not hinder the CVB3 PD-induced CPE. Full O- or N-desulfation (FDOS and DNS, respectively) of heparin results in a great loss of inhibitory potential against CVB3 PD. 6-O-desulfation (6ODS) of heparin leads to a loss of antiviral potential comparable to the loss after full O-desulfation, whereas 2-O-desulfated (2ODS) heparin still inhibits CVB3 PD-induced CPE, although to a lesser degree. The mean values and standard deviations of at least three separate experiments with two replicates each are shown.

The results from competition assays with specifically desulfated heparins indicate that fully de-O-sulfated heparin and de-N-sulfated heparin scarcely block virus replication (Fig. 1B). Like full de-O-sulfation and N-desulfation of heparin, 6-O-desulfation of heparin led to a strong loss of antiviral activity. In contrast, 2-O-desulfated heparin, which still contains 6-O-sulfates, exhibited a strong dose-dependent antiviral activity. Unmodified heparin inhibited the CVB3 PD-induced CPE to the greatest extent. The 50% inhibitory concentrations calculated from the mean dose-response curves were 12.5, >200, >200, 194.8, and 57.8 µg/ml for heparin and de-N-sulfated, full de-O-sulfated, 6-O-desulfated, and 2-O-desulfated heparin, respectively (Table 1). Taken together, these results confirm the crucial role of 6-O-sulfation of GlcN for interaction of cell surface HS with CVB3 PD and indicate that the disaccharide [IdUA-GlcNSO3(6OSO3)]n may be part of the binding sequence of HS for CVB3 PD.

Increase in endosomal pH affects viral infection, but low pH does not induce instability of CVB3 PD. CVB3 PD can utilize CAR as well as HS to enter into host cells. To examine whether CVB3 PD infection depends on a low endosomal pH, cell lines expressing only one of these two receptors (CHO-K1 cells and pgsD677-hCAR cells) were exposed to ammonium chloride. The weak base ammonium chloride penetrates acidic cell compartments, such as endosomes and lysosomes, and increases their pH. By using ammonium chloride, pH-dependent internalization, uncoating, or trafficking of viruses can be inhibited. After a 1-h pretreatment of cells with different concentrations of ammonium chloride in test medium, CVB3 PD was added for 3 h at room temperature. The cells were then washed three times with medium to remove both the compound and virus on the cell surface. Cell viability was scored spectrophotometrically after 2 days of incubation at 37°C. The viability of CVB3 PD-infected, ammonium chloride-treated CHO-K1 cells increased markedly in a dose-dependent manner (Fig. 2), with a 50% inhibitory concentration of 24.67 µM (Table 1). In contrast, an increase in cell viability was not observed after ammonium chloride treatment of pgsD-677-hCAR cells.


Figure 2
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  		FIG. 2. The lysosomotropic agent ammonium chloride inhibits CVB3 PD replication in CHO-K1 but not in pgsD-677-hCAR cells. Cells were preincubated with ammonium chloride for 1 h at 37°C. Virus was added, and incubation continued for 3 h at 37°C. The cells were then washed three times and incubated for another 2 days at 37°C. Afterwards, the cells were fixed and stained with a crystal violet solution in formalin and water. Cell viability was assessed spectrophotometrically after dye extraction. Two experiments with six replicates each per ammonium chloride concentration were carried out. The mean percentages of cell viability with standard deviations are shown.

Additionally, the inhibitory activities of the carboxylic ionophore monensin and of the proton ATPase inhibitor bafilomycin A1 were examined in HeLa, CHO-K1, and pgsD-677-hCAR cells. Both compounds were added to cells for 1 hour before virus infection and removed 3 h (HeLa cells) or 4 h (CHO-K1 and pgsD-677-hCAR cells) after virus addition, together with nonadsorbed virus. Incubation was continued for a further 4 h for HeLa cells, 20 h for pgsD-677-hCAR cells, and 40 h for CHO-K1 cells without compounds. Monensin (50 µM) inhibited CVB3 PD replication in HeLa cells but not in pgsD-hCAR cells (Fig. 3). The applied monensin concentration of 50 µM was very cytotoxic to CHO-K1 cells. Therefore, the results are not shown. CVB3 PD replication occurred in the presence of bafilomycin A1 in CHO-K1 and pgsD-677-hCAR cells (Fig. 3). A somewhat lower replication efficiency was observed in HeLa cells.


Figure 3
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  		FIG. 3. Effects of monensin, bafilomycin A1, chlorpromazine, sucrose, and nystatin on CVB3 PD antigen expression in HeLa, CHO-K1, and pgsD-677-hCAR cells. Cells were preincubated with inhibitors for 1 h at 37°C. CVB3 PD was added at an MOI of 10 TCID50/cell, and incubation was continued for another 3 or 4 h at 37°C. Afterwards, the cells were washed three times. Following a further 4 (HeLa cells), 20 (pgsD-677-hCAR cells), or 40 (CHO-K1 cells) hours of incubation at 37°C, cells were fixed, and viral antigen was detected with a monoclonal CVB3-specific antibody and APAAP staining. Three experiments with two or three replicates each per inhibitor were performed. Representative examples of photographs are shown.

Some picornaviruses, such as HRV2 and the HS-binding HRV89 variant, are unstable at low pHs (7, 37). Uncoating of these viruses can occur in the absence of specific receptors at the low pH in late endosomes. To test whether CVB3 PD is also unstable at low pHs, it was exposed to a low pH. Both CVB3 variant viruses were incubated in acetate buffer (pH 4.5) for 15 and 30 min at room temperature. Afterwards, the pH was brought back to neutrality. The resulting virus titers were compared to those at time zero. As shown in Table 2, both viruses were stable at low pH.


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  		TABLE 2. Sensitivity of CVB3 PD to low pH compared to that of CVB3 H3

Chlorpromazine, sucrose, and nystatin did not inhibit CVB3 PD infection. Endocytosis occurs by multiple mechanisms (9). Clathrin-coated vesicles or pits as well as caveolae can be used for endocytosis of viruses. Furthermore, clathrin- and caveola-independent endocytic pathways exist. Inhibitors of different endocytic pathways are widely used to gain insight into different endocytic pathways. In the present study, sucrose (0.3 mM) and chlorpromazine (10 µM), both of which inhibit the clathrin-dependent endocytic pathway, and nystatin (10 µg/ml), which belongs to the cholesterol-depleting drugs which can affect the caveola pathway, were examined. Surprisingly, neither sucrose and chlorpromazine (photographs of sucrose-treated cells are shown as an example) nor nystatin affected CVB3 PD antigen expression in HeLa, CHO-K1, and pgsD-677-hCAR cells when added 1 h before virus inoculation and during virus entry for a further 3 or 4 h (Fig. 3). Even if the compounds were present during the whole incubation time, no inhibitory effect was observed (results not shown).

A marked reduction in the number of CVB3 PD antigen-positive cells was detected after pretreatment of CHO-K1 but not pgsD-677-hCAR cells with the control compound heparin, which blocks viral attachment. Viral antigen expression was also affected, albeit at a low efficiency, in CVB3 PD-infected HeLa cells expressing HS as well as CAR.

CAR and HS usage by CVB3 PD has an effect on the time of virus attachment and penetration but does not influence the time required for a complete life cycle in Chinese hamster ovary cells with different receptor expression patterns. The times required for a complete multiplication cycle for picornaviruses generally range from 5 to 10 h (27). These viruses need no more than 1 to 2 h at 37°C to attach to and enter into permissive host cells, e.g., HeLa cells. After 1 hour, the inoculum can be removed and the cell monolayers washed three times without a marked loss of infectivity. But in studying the multiplication cycle of CVB3 PD in hCAR-negative CHO-K1 cells, a sharp loss of infectivity was observed if this experimental approach was used. The removal of virus-containing inoculum after 1 h of incubation at 37°C led to a nearly complete loss of infectivity. Therefore, comparative studies on the influence of the time of virus removal were performed, using the number of virus antigen-positive cells after CVB3 PD infection as a parameter for infectivity. To ensure synchronous infection, cells were infected at an MOI of 10. After an incubation time of 15 or 30 min or 1, 2, or 4 h at 4°C or 37°C, the virus inoculum was removed, and cells were washed three times with test medium. Viral antigen expression was examined 7, 24, and 48 h after virus inoculation into HeLa, pgsD-677-hCAR, and CHO-K1 cells, respectively. At these time points, maximum antigen expression was observed in corresponding virus controls (virus inoculum was not removed during the whole experimental time).

The following differences were found. As shown in Fig. 4A, infectivity was only weakly or not affected as a result of the removal of CVB3 PD-containing inoculum from hCAR-expressing HeLa and pgsD-677-hCAR cells after incubation for 1 h at 37°C. In contrast, the removal of virus-containing supernatant from CAR-negative, HS-expressing CHO-K1 cells markedly affected the number of CVB3 PD antigen-expressing cells until 2 h after virus inoculation. At least 4 h of virus incubation was necessary for effective infection of CHO-K1 cells at 37°C. If virus incubation was carried out at 4°C, a temperature permitting only binding, not viral entry, at least 2 hours of virus incubation was necessary to obtain maximum antigen expression in HeLa and pgsD-677-hCAR cells at the end of one multiplication cycle (results not shown). Viral antigen-positive cells were only sparse or not detected at all in CHO-K1 cell monolayers after virus attachment at 4°C (results not shown).


Figure 4
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  		FIG. 4. Differences in entry kinetics and multiplication cycle of CVB3 PD in HeLa, CHO-K1, and pgsD-677-hCAR cells. (A) The time necessary to initiate infection of CVB3 PD depends on receptor expression. Cells were infected at an MOI of 10 TCID50/cell. After the indicated incubation periods at 37°C, virus-containing supernatant was removed, cells were washed three times, and fresh medium was added. Seven (HeLa cells), 24 (pgsD-677-hCAR cells), or 48 (CHO-K1 cells) hours after virus infection, cells were fixed, and viral antigen was detected with a monoclonal CVB3-specific antibody by APAAP staining. The photographs show representative examples of at least three experiments with two replicates each per time point. (B) One-step growth experiments for CVB3 PD in HeLa, CHO-K1, and pgsD-677-hCAR cells. Virus was added at an MOI of 10 TCID50/cell. At the indicated times after virus inoculation, specimens of infected cells were frozen. Before titrating for infectivity in HeLa cells, the virus was released by three freeze-thaw cycles. Mean virus titers and standard deviations of three replicates per time point are shown.

The results from one-step growth experiments with CVB3 PD in the respective cell lines show that CVB3 PD requires approximately 6 h from adsorption to completion of virus assembly in HeLa cells (Fig. 4B). Virus titers began to increase 3 to 4 h after virus inoculation in both HeLa and pgsD-677-hCAR cells. In CHO-K1 cells, the first rise of viral titer was found 8 h after virus inoculation. In CHO-K1 as well as pgsD677-hCAR cells, maximum virus titers were observed 24 to 48 h after infection.


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DISCUSSION
 
Our previous studies showed that CVB3 PD has an extended cell tropism because amino acid substitutions in capsid protein VP1 enable it to use HSPG as an alternative receptor (29, 38). In this report, the modifications of the HS chain necessary for CVB3 PD binding were proven. Furthermore, the influence of several inhibitors of endocytic pathways on viral replication was examined. Additionally, the time necessary to initiate infection as well as the life cycle of CVB3 PD was comparatively studied in cell lines expressing only CAR or HSPG.

The resistance of pgsE-606 cells with a very low content of N-sulfates to CVB3 PD infection suggested that N-sulfated HS are a pivotal prerequisite for this virus to enter cells in the absence of CAR (38). In this study, this hypothesis was further confirmed by using competition assays with specifically desulfated heparins. This is a widely used approach to study the impact of specific sulfation of HS on virus-HS interactions (30). In strong contrast to heparin, N-desulfated heparin was nearly unable to hinder CVB3 PD infection of cells. This fact, in addition to findings obtained with the pgsE-606 cell line, prove that N-sulfation is not only a crucial step for further essential modifications of the HS chain which mediate the binding of CVB3 PD but is itself necessary for virus binding. N-sulfation of HS also plays an important role in attachment of herpes simplex viruses 1 and 2, papillomavirus, and respiratory syncytial virus (RSV) to their host cells (17, 19, 31). N-sulfated HS seems to be sufficient to mediate RSV infection (17). In contrast to the case for RSV, the results from antiviral tests with specifically desulfated heparins as well as heparan sulfate-binding growth factors in the present study demonstrate that besides N-sulfation, O-sulfation plays a crucial role in CVB3 PD entry. The plain loss of antiviral activity after full O-desulfation and selective 6-O-desulfation of heparin, but not after selective 2-O-desulfation of heparin, demonstrates that 6-O-sulfate-containing glucosamines are very important for the interaction of CVB3 PD with cell surface HS. Because 2-O-sulfate- as well as 3-O-sulfate-lacking, 6-O-sulfate-expressing pgsF-17 cells represent the cell line with the minimum of HS modifications for CVB3 PD susceptibility (38), a crucial role of 2-O-sulfate and 3-O-sulfate groups can be excluded. Obviously, the structural requirements of CVB3 PD for binding to HS differ from those of herpesviruses, which utilize 2-O-sulfated as well as 6-O-sulfated HS for attachment (14) and, additionally, need 3-O-sulfated moieties of HS for entry (21, 33).

To examine the role of the low pH of cellular vesicles in CVB3 PD infection, cells were treated with ammonium chloride, a weak base which increases the pH of endosomes and lysosomes. An increase of the low endosomal pH can inhibit viral internalization and cellular trafficking as well as viral uncoating. Ammonium chloride treatment inhibited the replication of CVB3 PD in CHO-K1 but not in pgsD-677-hCAR cells (Fig. 2), giving a hint that acidification of cellular vesicles is important for successful infection initiated by attachment to heparan sulfates. Moreover, monensin, which blocks endosomal acidification, also inhibited viral antigen expression in HeLa cells expressing CAR and HS but not in HS-negative pgsD-677-hCAR cells (Fig. 3). Unfortunately, this compound was very cytotoxic to CHO-K1 cells. Like HRV14 (4, 16), CVB3 PD replicated in the presence of the vacuolar H+-ATPase inhibitor bafilomycin A1, which prevents acidification of endosomes. Moreover, CVB3 PD was stable at a low pH (Table 1). Obviously, a low pH does not induce viral uncoating, as demonstrated, for example, for HRV2 and the HS-binding HRV89 variant (7, 37).

Endocytosis of viruses may occur via a clathrin- or caveolin-mediated pathway or independent from clathrin as well as caveolae. Clathrin-mediated endocytosis was described for the picornaviruses HRV2 (34) and HRV14 (16). Echovirus 1 (23), which also belongs to the picornavirus family, was shown to enter host cells by caveola-mediated endocytosis. Moreover, during preparation and revision of this report, two other scientific groups published their results on CVB3 entry pathways. Whereas Chung et al. (8) found that uptake of CVB3 H3 into HeLa cells depends on clathrin, Coyne and Bergelson demonstrated that CVB3 RD internalization into polarized CaCo-2 cells requires caveolin (10). Both groups used confocal microscopy to confirm the specific endocytic pathways. It is possible that the discovered distinct endocytic pathways depend on the cell type encountered and the receptors and coreceptors used by CVB3 PD, CVB3 H3, and CVB3 RD. CVB3 PD infection in HeLa, CHO-K1, and pgsD-677-hCAR cells was not affected by either inhibitors of the clathrin-dependent pathway (chlorpromazine and sucrose) or the cholesterol-depleting drug nystatin, which disturbs caveola integrity and hinders entry of these viruses. Therefore, neither of the two pathways could be confirmed by using inhibition experiments. Other methods that enable direct studies of the internalization of CVB3 PD and its colocalization with markers of endocytic pathways as well as experiments with dominant-negative inhibitors of components of the endocytic pathway are now ongoing to clarify the mechanism of entry.

While this report was being prepared, Vlasak et al. reported that HRV89 variants growing in cells deficient in intercellular adhesion molecule 1 (ICAM-1) also utilize HSPG as a cellular receptor and that a low pH prevailing in endosomal compartments is necessary for uncoating in the absence of the catalytic activity of ICAM-1 (37). Their data underscore the observation that picornaviruses can use HSPG as additional receptors and change their entry pathway. Like CVB3 PD, these HRV89 variants were obtained as the result of cell culture adaptation. Based on existing experience, the validity of the obtained conclusions can now be studied with naturally circulating CVB3.

Interestingly, CVB3 PD infection in CAR-expressing HeLa and pgsD-677-hCAR cells occurs much faster than that in CHO-K1 cells (Fig. 4A). The fast binding and uptake of this CVB3 variant by CAR are in strong agreement with recently published data (8, 10). If CVB3 PD attachment proceeds at 4°C, it binds somewhat slower to CAR-expressing cells. At 4°C, binding to HS-expressing, CAR-negative CHO-K1 cells is nearly abolished. However, the course of the viral life cycle was very similar in both Chinese hamster ovary cell lines and was markedly slower (approximately 24 h) (Fig. 4B) than that in HeLa cells (6 h) (Fig. 4B). CHO-K1 as well as pgsD-677-hCAR cells are hamster ovary cell lines differing only in receptor expression. Therefore, intracellular factors of hamster ovary cells rather than the usage of HS or CAR as the receptor seem to have an influence on the duration of the viral life cycle. A viral life cycle of 24 h was also found in CVB3-infected human fibroblasts (results not shown).

In summary, the results of the present study show that (i) specifically N- and 6-O-sulfated HS chains mediate attachment of CVB3 PD to hCAR-lacking cells, (ii) virus replication is dependent on a low endosomal pH, and (iii) the slower uptake of virions by HS than by CAR does not affect the life cycle duration in hamster ovary cells. The results of this study further prove not only that HS tether extracellular ligands such as growth factors and virions to the cell surface by nonspecific electrostatic interactions but that specifically sulfated HS also mediate endocytosis of virions into host cells.


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ACKNOWLEDGMENTS
 
We thank Markus Rene Lisy for excellent technical assistance.

This study was supported by grants from the DFG (SCHM 1594/1) and from Jenoptik, Jena, Germany.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Virology and Antiviral Therapy, Medical Centre of the Friedrich Schiller University Jena, Hans-Knoell-Str. 2, D-07740 Jena, Germany. Phone: 49 3641 657222. Fax: 49 3641 657202. E-mail: michaela.schmidtke{at}med.uni-jena.de. Back


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Journal of Virology, July 2006, p. 6629-6636, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.01988-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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