1 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Dep. Biotecnología, Ctra Coruña Km 7.5, 28040 Madrid, Spain
2 Serveis Cientificotècnics (Unitat de Citometria de Flux), Parc Científic de Barcelona, Spain
3 CIQ(UP)/Departamento de Química, Faculdade de Ciências da Universidade do Porto, P-4169-007 Porto, Portugal
4 Protein Design Group, Centro Nacional de Biotecnología, Cantoblanco, 28049 Madrid, Spain
Correspondence
Victoria Ley
ley{at}inia.es
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ABSTRACT |
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Present address: Biomolecular Structure and Modelling Unit, Biochemistry and Molecular Biology Department, University College London, Gower Street, London WC1E 6BT, UK.
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INTRODUCTION |
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Binding of viruses to HS is usually electrostatic in nature and of low specificity. Viral proteins typically bind to HS through positively charged amino acid residues, and in some instances the HS-binding domain has been characterized (Flynn & Ryan, 1996; Trybala et al., 1998
). Frequently, in addition to a low-affinity co-receptor that initiates the cell attachment, virus entry into the cell depends on high-affinity receptors (Chen et al., 1997
; Hung et al., 1999
; Qiu et al., 2000
). The role of HS as a co-receptor has been suggested in several viral infections. Hence, there are examples of viruses that can bypass the binding to HS by using other alternative receptors and co-receptors (Baranowski et al., 2000
).
Swine vesicular disease virus (SVDV) is a picornavirus of the enterovirus genus that causes an emerging disease of pigs (SVD) whose symptoms are similar to those caused by FMDV (Nardelli et al., 1968). The comparison of the complete genome sequences of SVDV and coxsackie B5 viruses (CVB5) reveals a close relationship between these two viruses (Zhang et al., 1999
). It has been demonstrated that SVDV is a subspecies of human CVB5 that arose as a result of an adaptation to swine. The divergence from a common ancestor has been estimated by phylogenetic studies to have occurred between 1945 and 1965 (Zhang et al., 1999
).
The initial events in the cycle of SVDV infection are not yet well characterized. It has been recently shown that, as in the case of coxsackievirus B1-6, the coxsackievirus-adenovirus receptor (CAR) is a functional receptor for SVDV (Martino et al., 2000). Also, the decay-accelerating factor (DAF), used as co-receptor for coxsackievirus A21, B1, B3, B5, echovirus 6, 7, 11, and enterovirus 70 appears to have a role in SVDV entry into cells (Martino et al., 2000
). However, it has been shown that HS can be used as an alternative receptor for some picornaviruses, such as FMDV (Baranowski et al., 2000
) and CVB3 (PD strain) (Zautner et al., 2003
), in some conditions or when the classic receptor is absent, a fact that supports the flexibility in picornavirus receptor usage. To investigate whether HS plays a role in SVDV infection, we have analysed the interaction of SVDV with HS and other glycosaminoglycans (GAGs).
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METHODS |
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Nucleotide sequence analysis of SVDV.
Viral RNA was extracted by standard techniques and subjected to RT-PCR using the SuperScriptII kit (Gibco-BRL) following the manufacturer's protocol. The oligonucleotides used for the RT-PCR are shown in Table 1. PCR products were purified with the Wizard PCR Preps DNA purification system (Promega) and sequenced at the Departamento de Secuenciación, Centro de Investigaciones Biológicas (CIB-CSIC, Spain).
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Inhibition of infection assay.
IB-RS-2 cells (104 cells per well) were infected with 104 p.f.u. per well of SVDV in the presence of duplicate dilutions of each soluble inhibitor (heparin/HS/CS-A, -B, -C, PF4) at 37 °C for 20 h. The inhibition of the CPE observed was quantified as above and expressed as percentage cell survival. To further study the effect caused by heparin, we performed the inhibition of infection assay in different conditions: (1) virus was incubated with heparin for 30 min at 37 °C prior to addition to cell cultures; (2) heparin was added to cultures of virus-adsorbed cells (cells were first incubated with SVDV at 4 °C for 30 min and washed with cold DMEM) and incubated for 1 h at 37 °C; and (3) cells were incubated with heparin for 30 min at 37 °C and washed before addition of the virus. All these experiments were performed using 100 p.f.u. SVDV per well. Cells were washed with medium at 1 h post-infection to remove non-adsorbed virus and cultures were incubated overnight at 37 °C. The inhibition of the CPE was determined as above.
SVDV infection of IB-RS-2 cells treated with heparinase I or III.
Confluent monolayers of IB-RS-2 cells in 12-well plates were washed twice with DMEM and incubated with 0·2 ml 1·5x10-2 U ml-1 or 3x10-2 U ml-1 of heparinase I or with 0·5x10-2 U ml-1 or 10-2 U ml-1 of heparinase III for 1 h at 37 °C with gentle shaking. The cells were washed twice with DMEM and approximately 50 p.f.u. of SVDV in 0·2 ml DMEM were added. Following virus adsorption for 1 h at 37 °C with gentle shaking, the cells were washed twice with medium and overlaid with DMEM supplemented with 10 % FCS and containing 0·6 % agarose. The plaques were detected by staining cells with crystal violet solution after 30 h incubation at 37 °C.
Selection of SVDV variants with lost affinity for heparin.
SVDV variants (heparin-resistant variants, Hepres) with lost affinity for heparin were selected by serial rounds of infection in the presence of increasing inhibitory concentrations of soluble heparin, following the method described for the selection of SVDV monoclonal antibody neutralization-resistant (MAR) mutants (Borrego et al., 2002). SVDV (5x106 p.f.u. ml-1) was seeded in 96-well plates over IBRS-2 cells and in the presence of heparin. Three rounds of selectionamplification were repeated, increasing the heparin concentration up to 1·5 mg ml-1. Seven wells showing cell lysis in the presence of heparin were obtained in the last round. These SVDV variants were isolated and amplified in the presence of heparin for further analysis.
Location of mutations affecting heparin binding.
The amino acid substitutions found in SVDV variants lacking the heparin-binding phenotype were initially mapped using an SVDV homology model (Jiménez-Clavero et al., 2000). The crystal structure of SVDV recently determined (Jiménez-Clavero et al., 2003
; Verdaguer et al., 2003
) confirmed the location of these amino acid substitutions.
Surface plasmon resonance (SPR) analysis.
Preparation of surfaces of sensor chips.
HS was biotinylated as described (Lookene et al., 1996) and immobilized on avidin bound to the surface of the sensor chip by injecting a solution of HS-biotin (100 µg ml-1 in HBS buffer: 10 mM HEPES; 0·15 M NaCl; 3·4 mM EDTA and 0·005 % surfactant P20; pH 7·2) at 5 µl min-1 continuous flow. The CM-5 sensor chip carboxymethyl surface was activated by a 7 min injection of 0·2 M EDC (N-ethyl-N'-dimethylaminopropylcarbodiimide), 0·05 M NHS (N-hydroxysuccinimide). Finally, avidin (100 µg ml-1 in 10 mM sodium acetate buffer pH 5·5) and biotin (100 µg ml-1 in HBS) were consecutively injected. Surfaces were then regenerated with 0·5 M NaCl in 10 mM NaOH, and the final two-step immobilization levels were of 2·5 ng avidin mm2 and 0·2 ng HS-biotin mm2, respectively.
Solutions.
Serial dilutions of virus (411320 µg protein ml-1) were prepared in HBS buffer. In samples for BIAcore analysis, heparin (1 mg ml-1 in HBS) was used to dissociate the virusHS complexes, and 1 M NaCl to regenerate the HS surfaces. Heparin solutions (0·06216 µg ml-1 in HBS) were employed in solution-affinity SPR experiments. The BIAcore 1000 instrument, the sensor chip CM-5, commercial HBS buffer, amine coupling kit NHS, EDC and ethanolamine were purchased from Biosensor AB.
Direct binding assays.
All direct SPR analyses were run at a 5 µl min-1 HBS flow and each virus preparation was injected at six different concentrations, ranging from 41 to 1320 µg protein ml-1. Sensorgrams were generated by injections of virus solutions using 2 min association steps followed by 3 min dissociation in heparin (1 mg ml-1) (co-injection mode). After an additional 3 min dissociation step in running buffer, the surfaces were regenerated by a 2 min pulse of 1 M NaCl without any measurable loss in baseline level or binding activity of the surface. Biosensor data were prepared, modelled and fitted using BIAevaluation 3.0.2 software (O'Shannessy et al., 1993), and global curve fitting was done by non-linear least-squares analysis (Morton et al., 1995
) applied simultaneously to the entire dataset. The quality of the fitted data was evaluated by visual comparison between calculated and experimental curves as well as by the magnitudes of the chi-squared parameter. Initial binding rates were also measured from the linear slope of the sensorgrams at the initial stage of the association step (15 s after injection plug).
Competitive SPR assays.
Calibration curves of initial binding rate vs virus concentration were built from data obtained in the direct SPR assay above and fitted to a four-parameter equation (when possible) using the BIAevaluation 3.0.2 software. This equation was then used to calculate free virus concentrations in solution affinity assays. Virusheparin interactions were determined by overnight incubation of different heparin concentrations (0·06216 µg ml-1) at 4 °C, with a constant virus concentration (660 µg protein ml-1) in HBS. Virusheparin mixtures were placed at 25 °C prior to injection on the HS surface for virus quantification. The amount of virus remaining free to bind the immobilized HS was quantified by measuring initial binding rates and extracting the corresponding virus concentrations from the relevant calibration curves. The variations of free virus with heparin concentration were plotted as inhibition curves.
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RESULTS |
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As shown in Table 2, the infection was completely inhibited, in a dose-dependent manner, in experiment (1), whereas no inhibition was observed in experiments (2) and (3). These results indicated that the heparin-mediated inhibition of SVDV infection was caused by a direct interaction with the virus, and that the interaction of heparin with the cells, if it occurs, had no effect on SVDV infection. Moreover, they showed that the presence of heparin inhibits the attachment of the virus to the cell surface, but once the virus is allowed to bind to the cell, the addition of heparin has no effect on the infection. Taken together, these results suggest that the effect of heparin on SVDV infection is a result of a competitive inhibition, hampering SVDV interaction with cell surface GAGs analogous to heparin, such as HS proteoglycan. To confirm this point, we carried out in vitro SVDV infections in the presence of different dilutions of soluble HS or heparin. As shown in Fig. 2(A)
, both HS and heparin inhibited SVDV infection, with HS requiring concentrations nine times higher than heparin (0·9 mg ml-1 vs 0·1 mg ml-1) to obtain the same inhibitory effect. As these GAGs have the same basic sugar chain structure but differ in their degree of sulphation, it seemed likely that the differences in the inhibition potency observed between them were related to this characteristic. To assess this hypothesis, we performed the same assay using desulphated heparin in parallel with normal heparin. Irrelevant (<20 % cell survival) inhibition was observed when adding up to 300 µg ml-1 of desulphated heparin, whereas a typical inhibition curve (>90 % cell survival at 40 µg ml-1) was observed in the presence of normal heparin (Fig. 2B
). To further analyse the effect of different GAGs on the inhibition of SVDV infection, we carried out the same inhibition assay in the presence of chondroitin sulphate (CS)-A, CS-B (also known as dermatan sulphate) and CS-C. All of them are sulphated, but in different positions and/or with different sugar chain constituents. Besides heparin, only CS-B (dermatan sulphate) caused a relevant inhibition of the SVDV infection, similar to that obtained with HS. Neither CS-A nor CS-C showed any effect on SVDV infection at the concentrations tested (Fig. 2C
). To further determine the physiological relevance of cell surface HS for SVDV binding we studied the effect of platelet factor 4 (PF4), a small basic growth factor that binds to heparin and to cell surface HS, on SVDV infection. As shown in Fig. 2(D)
, the infection was strongly inhibited in the presence of increasing amounts of PF4, indicating that cell surface glycoconjugates such as HS were needed for SVDV infection in vitro in IB-RS-2 cells.
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Phenotypic selection of variants not inhibited by heparin was carried out by infecting IB-RS-2 cells with SVDV in the presence of increasing concentrations of soluble heparin, as described in Methods. After three rounds of selection the last of them in the presence of 3·25 mg heparin ml-1 seven SVDV variants showing a complete lack of susceptibility to inhibition by soluble heparin (Hepres phenotype) were isolated. Nucleotide sequence analysis of the structural (P1) region of the genome of each of the seven variants revealed that they were identical in this region; thus we considered them as being the same viral variant, perhaps present in the original virus population in undetectable amounts. Comparison of nucleotide-derived amino acid sequences obtained from the parental isolate and the Hepres variant revealed two amino acid substitutions in the capsid: one conservative (A/V) at position 135 of VP2 (A2135V) and one non-conservative (I/K) at position 266 of VP1 (I1266K). Multiple sequence alignments showed that there was no overall conservation of the region around these two positions in the Picornaviridae (Fig. 4).
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DISCUSSION |
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The assays used to investigate the mechanism of the HSvirus interaction indicated that the interaction of SVDV with cellular HS occurs during cell attachment, but once the virus is bound, addition of HS does not affect the process of infection. Therefore, as proposed for other viruses, the binding of SVDV to cellular GAGs probably mediates an early step of the viruscell interaction, facilitating the subsequent recognition of other receptors such as CAR (Martino et al., 2000). However, it cannot be ruled out that HS is being used as an alternative receptor. Inhibition of viral infection by soluble heparin is a conventional approach to determine the ability to bind to heparin by many viruses. It has been recently published that among echoviruses (EV), the species most sensitive to the inhibition by heparin are EV 9 and EV 25, requiring 125 µg ml-1 to inhibit the infection. In contrast, some EV such as 1, 4, 12 and 13, as well as other enteroviruses like PV3, CVB2 and CVB3, are insensitive to heparin up to 2 mg ml-1 (Goodfellow et al., 2001
). Using the same method, SVDV appears to be especially sensitive to inhibition by heparin, since only 100 µg ml-1 completely inhibited the infection of IB-RS-2 cells in comparable conditions.
The role of cell surface GAGs in SVDV attachment was further assessed by treating IB-RS-2 cells with heparinase I and III prior to the incubation with the virus. In these experiments, the infectivity of SVDV was strongly reduced after treatment with heparinase III, whose principal substrate is HS, and to a lesser extent with heparinase I, whose substrate is heparin. Our data therefore support the hypothesis that SVDV interacts with cell surface HS during the process of infection.
It has been shown that HS interactions with proteins are mostly electrostatic in nature, the positively charged amino acid residues interacting with the negatively charged sulphates (Fromm et al., 1995). This is probably the case for HSSVDV interaction, since there was a good correlation between degree of sulphation and inhibition of infection. Thus, the most effective substrate for binding was heparin, which is the most sulphated, followed by HS and dermatan sulphate, while chondroitin sulphates A and C were less effective. In addition, the failure of SVDV to bind desulphated heparin corroborated this hypothesis. However, the carbohydrate backbone of the GAG may add specificity to the interaction, resulting in the relatively high affinity of the SVDVHS interaction observed in the SPR analysis.
The rapid appearance of heparin resistant (Hepres) variants, after three passages in heparin-containing cultures, suggests either a high rate of the mutation(s) responsible for this phenotype, or alternatively, the presence of a minor population of a viral variant resistant to heparin, which was not detected in the heparin-Sepharose chromatography experiment. The results of sequence analysis of the seven Hepres isolates, showing that all of them had the same two substitutions in the VP1 and VP2 proteins, respectively, suggest that they might be the same SVDV variant, present in undetectable amounts in the original population. The analysis of the kinetics of virusheparin binding by SPR showed that the association and dissociation rate values of the parental SVDV corresponded to a high-affinity interaction. In contrast, the sensorgrams corresponding to the seven Hepres isolates were similar and indicated a lack of interaction with heparin. Analysis of the amino acid sequences of the P1 structural polyprotein of the Hepres variant revealed that the two substitutions in VP1 and VP2 proteins were located in the same region on the 3D model of the SVDV protomer, defining a potential HS-binding domain that is well exposed on the viral capsid (Fig. 6). Interestingly, this region almost overlaps with a cluster of amino acids that are changed between old and recent SVDV isolates, and located in the neighbourhood of, but not overlapping, the CAR footprint (Fry et al., 2003
). The region homologous to this cluster in the echovirus 7 capsid has been implicated in DAF binding (He et al., 2002
). Pig DAF contains homologues for only three out of the four SCRs that are encoded by human, rat and mouse DAF. Sequence comparison indicates that it is the fourth SCR that is missing. Moreover, the accumulation of changes in the putative DAF-binding site in the recent SVDV isolates suggests adaptive modifications to a different interaction, perhaps with the pig homologue of human DAF, or with a porcine molecule still not identified. In this context, cell surface HS may be a good candidate. Whether HS substitutes DAF as the attachment receptor in the pig, or participates in the cell binding as an alternative receptor, remains to be studied.
The loss of heparin binding by the Hepres variant is not straightforward, based on the electrostatic charge of the amino acid substitutions. There is a conservative substitution A2135V in the VP2 protein and a non-conservative one I1266K in the VP1 that does not imply the loss of a positively charged amino acid. The two substitutions are close to each other and adjacent to a region of relatively high concentration of basic amino acids, characteristic of the heparin-binding domains of proteins. Three lysines, K1253, K1258 and K1259, at the C terminus of VP1 and one arginine R3073 in the BC loop of VP3 contributed to this region. The crystal structure of FMDV serotype O in complex with HS (Fry et al., 1999) showed the HS binding site of the virus in a depression of positive electrostatic charge on the capsid, contributed by the three surface proteins: VP1, VP2 and VP3. The predicted HS binding for SVDV maps near, but does not overlap, the FMDV HS binding site (Fry et al., 1999
; Verdaguer et al., 2003
). Among the two amino acid substitutions that seem to be involved in the HS binding site, position 1266 is relatively variable among other SVDV variants. However amino acid 2135 is conserved among old and recent SVDV isolates, including CVB5 (Verdaguer et al., 2003
). As indicated above, CVB5 also binds HS; therefore it seems that 2135 might be more important than 1266 for maintaining the HS binding site functional. The most likely explanation for these findings is that this basic domain is involved in the binding to HS, and that slight differences in the amino acid sequences in an adjacent region might produce a strong difference in binding activity. Taken together, these studies indicate a role for GAGs in SVDV attachment: the virus probably binds highly sulphated forms of cell surface HS, which is likely to provide the initial interaction step during SVDV infection.
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ACKNOWLEDGEMENTS |
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Received 26 August 2003;
accepted 18 November 2003.