Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin {alpha}5{beta}1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding

Terry Jackson1, Wendy Blakemore1, John W. I. Newman1, Nick J. Knowles1, A. Paul Mould2, Martin J. Humphries2 and Andrew M. Q. King1

Department of Molecular Biology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK1
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK2

Author for correspondence: Terry Jackson. Fax +44 1483 237161. e-mail terry.jackson{at}bbsrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, {alpha}v{beta}3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin {alpha}5{beta}1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to {alpha}5{beta}1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins {alpha}5{beta}1 and {alpha}v{beta}3 and evidence is provided that {alpha}5{beta}1 might be an important FMDV receptor in vivo.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and-mouth disease virus (FMDV) is a member of the Picornaviridae, a family of small, non-enveloped, positive-stranded RNA viruses containing many important pathogens of humans and animals (Belsham, 1993 ). The virus exists in seven serotypes, types O, A and C, Asia-1 and the South African Territories (SAT) types 1, 2 and 3, each composed of numerous subtypes. A major structural feature of the outer capsid surface of the virion is a long, conformationally flexible loop (Acharya et al., 1989 ; Logan et al., 1993 ; Lea et al., 1994 ; Curry et al., 1996 ; Fry et al., 1999 ), the GH loop of VP1, which includes a highly conserved Arg–Gly–Asp (RGD) motif at its apex. Two classes of cell-surface receptors have been identified for FMDV, namely heparan sulphate proteoglycans (HSPG) and integrins. Field isolates of FMDV use RGD-dependent integrins as receptors for their internalization, through an interaction mediated by the GH loop RGD motif (Mason et al., 1994 ; Berinstein et al., 1995 ; Jackson et al., 1997 ). By contrast, strains of FMDV that have been multiply passaged through cultured cell lines acquire a high affinity for heparan sulphate and, as a consequence, an apparent ability to use HSPG as receptors for both attachment and internalization into cells without the mediation of integrins (Jackson et al., 1996 ; Sa-Carvalho et al., 1997 ; Neff et al., 1998 ).

Integrins are a family of transmembrane {alpha}/{beta} heterodimeric glycoproteins that are responsible for a variety of processes, including adhesion both between cells and between cells and the extracellular matrix and induction of signal transduction pathways that modulate various processes including cell proliferation, morphology, migration and apoptosis (Hynes, 1992 ; Montgomery et al., 1994 ; Springer, 1990 ). A general property of integrins, like all receptors, is that they exist in active (competent to bind ligand) or inactive (unable to bind ligand) states (Springer, 1990 ). Currently, the conversion from an inactive to an active state (integrin activation) is postulated to occur through two different mechanisms, collectively referred to as ‘inside-out signalling’; firstly avidity modulation, by clustering of heterodimers within the plane of the membrane, and secondly affinity modulation, mediated through conformational changes in the integrin ectodomain. At present, the molecular mechanisms that regulate these processes in vivo remain unclear; however, both processes require the cytoplasmic domains of the integrin and cellular proteins (Zhang et al., 1996 ; Dedhar & Hannigan, 1996 ; O’Toole et al., 1994 , 1995 ; Kashiwagi et al., 1997 ; Hughes et al., 1997 ). Fortunately, the conformational changes that occur naturally in the extracellular domains upon activation can be induced experimentally by reagents that act directly on the integrin, such as manganese ions or activating monoclonal antibodies. Manganese ions are believed to stabilize shapes of the ligand-binding pocket that favour ligand binding (Lee et al., 1995 ; Li et al., 1998 ) and activating antibodies enhance binding by stabilizing epitopes that are expressed only on the active conformation of the integrin (Bazzoni et al., 1995 ; Mould et al., 1995a ).

The primary route of infection by FMDV is believed to be through the pharynx, although it is not known whether virus replication takes place initially in epithelial or lymphoid cells (Salt, 1998 ). Currently, {alpha}v{beta}3 is the only integrin that has been shown to act as a receptor for internalization of FMDV and this is mediated by the FMDV RGD sequence (Berinstein et al., 1995 ). Several other integrins, including {alpha}v{beta}1, {alpha}v{beta}5, {alpha}v{beta}6 and {alpha}5{beta}1, bind their ligands through recognition of an RGD motif and these integrins are expressed on epithelial and lymphoid cells (Springer, 1990 ; Mette et al., 1993 ; Damjanovich et al., 1992 ), where they could serve as cellular receptors for FMDV.

Binding of FMDV to specific integrins on the surface of cells is complicated by the ability of some FMDV strains to bind heparan sulphate, which is usually present in a vast molar excess over integrins, and by the presence on most cells of many RGD-dependent integrin species (Jackson et al., 1996 ). Therefore, in order to characterize the molecular interactions between FMDV and integrins, we have adopted a strategy of using integrins in a purified form. By using this approach, we have shown previously that binding of FMDV to {alpha}v{beta}3 mimics the binding of its natural ligand, vitronectin, in that virus binding requires divalent cations, is maximal in the presence of manganese ions and can be inhibited specifically by low concentrations of RGD peptides (Jackson et al., 1997 ). In this study, we have sought to determine whether FMDV, in the absence of heparan sulphate and other integrins, can function as a ligand for the integrin {alpha}5{beta}1, which has fibronectin (Fn) as its natural ligand (Pierschbacher & Ruoslahti, 1984 ; Pytela et al., 1985 ). Previous studies have suggested that the GH loop of VP1, when expressed either as a fusion protein with {beta}-galactosidase or on the surface of hepatitis B virus core particles, functions as a ligand for {alpha}5{beta}1 (Villaverde et al., 1996 ; Chambers et al., 1996 ). This latter study also reported that viruses representative of some FMDV serotypes demonstrated weak binding to a purified preparation of {beta}1 integrins that had been enriched for {alpha}5{beta}1 (Chambers et al., 1996 ). In this report, we have extended these studies and show that FMDV behaves as an authentic RGD-dependent ligand for {alpha}5{beta}1. In addition, we show that the identity of the amino acid immediately following the GH loop RGD motif influences dramatically the ability of FMDV to bind integrins {alpha}5{beta}1 and {alpha}v{beta}3. The data are discussed in relation to the GH loop sequence of naturally occurring field isolates of FMDV.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Protein purification.
Virus purification on sucrose gradients from infected BHK cells has been described previously in detail (Curry et al., 1992 ). Trypsin-treated O1BFS was prepared by digesting gradient-purified virus with 200 pg/ml trypsin for 60 min at 25 °C. The enzyme was then inactivated by the addition of soybean trypsin/chymotrypsin inhibitor (400 pg/ml) and the virus was repurified on a second sucrose gradient. Purification of the human integrins {alpha}5{beta}1 and {alpha}4{beta}1 was done as described previously (Mould et al., 1995b , 1996 ).

{blacksquare} Viruses, antibodies and peptides.
The viruses used in this study were O1BFS (Logan et al., 1993 ), O1K-car2 (Kitson et al., 1990 ), O1K-f480 (Crowther et al., 1993 ), C-S8c1 (Lea et al., 1994 ), SAT-1 Bot-1/68 (N. J. Knowles & A. R. Samuel, unpublished data), SAT-2 Rho 1/48 (Rowe, 1993 ) and SAT-3 Zim 4/81 (B. E. Clarke, unpublished). FMDV O1BFS and SAT-1 Bot-1/68 bind heparin/heparan sulphate (Jackson et al., 1997 ; and unpublished observations), whereas the other viruses used in this study do not appear to bind heparin in vitro and are believed to use RGD-dependent integrins as cellular receptors without the mediation of HSPG (Baranowski et al., 1998 ; and unpublished observations). The anti-integrin antibodies used in these studies were the activating anti-{beta}1 antibody 9EG7 (Bazzoni et al., 1995 ) and the inhibitory antibodies P4C10 (anti-{beta}1, BRL) and JBS5 (anti-{alpha}5, Serotec). The GRGDSP and GRGESP peptides were purchased from Novabiochem. The O1BFS VP1 GH loop peptide (141VPNLRGDLQVLA152) and the control RGE version were synthesized on the peptide synthesis facility at the Oxford Centre for Molecular Science (New Chemistry Laboratory, Oxford, UK).

{blacksquare} Solid-phase binding assay.
The standard assay was carried out as follows. Plastic 96-well plates were coated with integrin {alpha}5{beta}1 (~1 µg/ml) in coating buffer (25 mM Tris–HCl, pH 7·4, 150 mM NaCl, 1 mM CaCl2, 0·5 mM MgCl2 and 1 mM MnCl2) for 16 h at 4 °C and the wells were blocked for 2 h with 200 µl binding buffer [25 mM Tris–HCl, pH 7·4, 150 mM NaCl, 1 mM MnCl2 3% radioimmunoassay grade BSA (ICN)]. Virus, in binding buffer, was added to the wells for 2–3 h at room temperature. The wells were washed with wash buffer (binding buffer without BSA) and bound virus was detected by using either a guinea pig or rabbit anti-FMDV serotype-specific polyclonal antiserum or anti-type O monoclonal antibodies, followed by a rabbit anti-guinea pig, goat anti-rabbit or goat anti-mouse alkaline phosphate conjugate (Sigma). For competition with peptides or experiments using antibodies, a 2x concentrated stock of virus was mixed with an equal volume of 2x concentrated peptide or antibody before addition to the immobilized integrin in 96-well plates, prepared and blocked as above. To determine the cation dependence of virus binding, 96-well plates, prepared and blocked as above, were washed twice with 5 mM EDTA [prepared in cation-free Tris-buffered saline (TBS)] for 2 min with a further wash for 5 min. The wells were then washed and blocked for a further 1 h and virus was bound in TBS–3% BSA containing the appropriate cation.

Integrin {alpha}v{beta}3 (Chemicon) was coated at 0·5 µg/ml in coating buffer (25 mM Tris–HCl, pH 7·4, 150 mM NaCl, 1 mM CaCl2, 0·5 mM MgCl2) for 16 h at 4 °C and the wells were blocked for 2 h with 200 µl binding buffer (25 mM Tris–HCl, pH 7·4, 150 mM NaCl, 3% BSA, 1 mM CaCl2, 0·5 mM MgCl2, 1 mM MnCl2). Virus was added to the wells for 2 h at room temperature. The wells were washed with wash buffer (binding buffer without BSA) and bound virus was detected as for {alpha}5{beta}1.

{blacksquare} Direct sequencing of viral RNA from epithelial tissue.
Direct sequencing of viral RNA from epithelial tissue was carried out as described previously (Knowles & Samuel, 1994 ). Briefly, viral RNA was extracted by QIAamp spin columns (Qiagen) or by phenol–chloroform extraction followed by ethanol precipitation from a 10% epithelial suspension derived from infected animals. Antisense oligonucleotide primers, complementary to the 2B region of FMDV, were used to prime a reverse transcriptase reaction. The reaction products were used as a template for a PCR with Taq DNA polymerase (Promega) and primers that amplify VP1 specifically. Thereafter, residual oligonucleotide primers were removed by using a Wizard PCR Preps kit (Promega) according to the manufacturer’s instructions and the GH loop region of VP1 was sequenced by using a internal Cy5-labelled primer and an fmol cycle DNA sequencing kit (Promega). Sequencing reactions were run on an ALFexpress DNA analysis system.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
FMDV is a ligand for the high-affinity conformation of {alpha}5{beta}1
Fn binding to {alpha}5{beta}1 is regulated differentially by divalent cations. Maximal binding occurs in the presence of manganese, whereas magnesium supports binding to a lower level and binding does not occur in the presence of calcium (Mould et al., 1995b ). We therefore examined the ability of divalent cations to support binding of FMDV to {alpha}5{beta}1 (Fig. 1a). We found that, as for Fn, FMDV binding to {alpha}5{beta}1 was dependent on divalent cations, as binding did not occur in the presence of 10 mM EDTA, and that binding was maximal in the presence of manganese ions but did not occur in the presence of calcium. However, magnesium at 1 mM failed to support virus binding. Increasing the cation concentration to 10 mM permitted intermediate levels of binding in magnesium, whereas virus binding could not be detected in the presence of calcium (data not shown). In the presence of manganese ions, viruses representative of several FMDV serotypes were found to bind to {alpha}5{beta}1 in a concentration- dependent manner (Fig. 1b). However, the SAT-2 virus appeared to be a poor ligand for {alpha}5{beta}1. Under identical assay conditions, we were unable to detect significant binding of O1BFS or SAT-3 to {alpha}4{beta}1, demonstrating that FMDV binding was specific for {alpha}5{beta}1 and not a general property of {beta}1 integrins. In addition, pre-treatment of immobilized {alpha}5{beta}1 with antibodies directed against either the {alpha}5 chain (JBS5, 10 µg/ml) or the {beta}1 chain (1:100 dilution of PC410) reduced binding of SAT-3 (5 µg/ml) by more than 95%, whereas a control antibody had no effect at 10 µg/ml (data not shown).



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Fig. 1. Cation dependence of FMDV binding to {alpha}5{beta}1 in a solid-phase assay. (a) Virus (SAT-3 at 10 µg/ml) was bound to immobilized integrin in the presence of either 10 mM EDTA or 1 mM divalent cation. Background (bg) was measured by omitting virus from the wells. (b) Binding of viruses representative of several FMDV serotypes to {alpha}5{beta}1 in the presence of 1 mM manganese ions. The viruses used are as indicated under the bars. tt, Trypsin-treated O1BFS. For both (a) and (b), integrin-bound virus was detected by using an anti-serotype-specific polyclonal serum and each data point is the mean of duplicate wells. The experiment was performed three times, each giving similar results.

 
The data shown in Fig. 1 suggested that the activation state of the integrin may be an important determinant of the ability of FMDV to bind {alpha}5{beta}1. Since Fn binding to {alpha}5{beta}1 in manganese can be further enhanced by the addition of activating antibodies (Mould et al., 1996 ), we examined the effects on virus binding of one such antibody, 9EG7. Fig. 2 shows that, in the presence of 9EG7, FMDV binding to {alpha}5{beta}1 was enhanced over that in manganese alone, confirming that the activation state of the integrin determines the ability of FMDV to bind this integrin. Binding of the SAT-2 virus was also enhanced in the presence of 9EG7, although this virus still appeared to be a poor ligand for {alpha}5{beta}1 when compared with the SAT-3 and O1BFS viruses.



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Fig. 2. Binding of FMDV to {alpha}5{beta}1 in the presence of manganese is further enhanced by the activating anti-{beta}1 antibody 9EG7. FMDV (as indicated) at the concentration indicated was bound to {alpha}5{beta}1 in the presence (filled bars) or absence (open bars) of activating anti-{beta}1 monoclonal antibody 9EG7 (10 µg/ml). Viruses were detected by using an anti-serotype-specific polyclonal serum. Each data point is the mean of duplicate wells. The experiment was performed four times, each giving similar results. Note that binding was enhanced for all three viruses tested.

 
FMDV does not bind {alpha}5{beta}1 in a buffer containing calcium and magnesium (Fig. 1). In an infected animal, FMDV would be required to enter cells in an environment that contained physiological concentrations of these cations. We therefore sought to determine whether the lack of binding of virus to {alpha}5{beta}1 in the presence of calcium and magnesium could be overridden upon activation of the integrin by 9EG7. Fig. 3 shows that, upon activation by 9EG7, FMDV is a ligand for {alpha}5{beta}1 in a buffer that contains both calcium and magnesium.



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Fig. 3. Binding of FMDV to {alpha}5{beta}1 in the presence of calcium and magnesium is stimulated by the activating anti-{beta}1 antibody 9EG7. FMDV (O1BFS at 10 µg/ml) was bound to {alpha}5{beta}1 in TBS–3% BSA containing 1 mM calcium and 1 mM magnesium in the presence (filled bar) or absence (open bar) of the activating anti-{beta}1 monoclonal antibody 9EG7 (10 µg/ml). Non-specific binding (hatched bar) was measured by binding virus in the presence of 9EG7 (10 µg/ml) and 2 µM GRGDSP peptide. Virus was detected by using an anti-type O guinea pig polyclonal serum. Each data point is the mean of triplicate wells. The experiment was performed twice, each giving similar results.

 
Binding of FMDV to purified {alpha}5{beta}1 is RGD-dependent
Enzymatic cleavage of FMDV strain O1BFS with trypsin removes both the C terminus (residues 201–213) and the GH loop (residues 139–154) of VP1, the latter cleavage resulting in the loss of the RGD motif (Strohmaier et al., 1982 ). Fig. 1(b) shows that trypsin treatment of O1BFS rendered the virion incapable of binding {alpha}5{beta}1, which suggests that the GH loop RGD motif is important for binding to {alpha}5{beta}1. RGD-dependent binding of ligands to integrins can be inhibited by peptides containing an RGD motif (Pytela et al., 1986 ). Fig. 4 shows that a GRGDSP peptide competed for binding of FMDV to {alpha}5{beta}1 in a concentration-dependent manner, reaching a maximal effect of ~95% inhibition. The effect of this peptide was specific, as an RGE version (GRGESP) had a minimal effect on virus binding at a concentration of 1 µM (data not shown). Table 1 shows that the amount of the GRGDSP peptide required to compete for binding by 50% (IC50) was similar for all the viruses tested. An accurate estimate of the IC50 for the SAT-2 virus could not be obtained due to the low level of binding in the absence of peptide (see Figs 1 and 2). In the presence of the activating antibody, 9EG7, the GRGDSP peptide was found to be equally as effective at inhibiting binding of FMDV O1BFS to {alpha}5{beta}1, indicating that, after activation by 9EG7, the initial interaction between FMDV and the integrin is still mediated by the RGD motif. A longer RGD-containing peptide (VPNLRGDLQVLA), with its sequence derived from the GH loop of VP1 of O1BFS, was also found to inhibit binding of O1BFS to {alpha}5{beta}1 specifically (the RGE version of this peptide had a minimal effect at the higher concentrations), but this peptide was found to be a less potent inhibitor (IC50 77±33 nM, n=3) of virus binding than the shorter GRGDSP peptide (Table 1).



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Fig. 4. Peptides that contain an RGD motif inhibit {alpha}5{beta}1–virus interactions. The percentage of bound virus is plotted against the concentration of competitor peptide. Continuous lines are the GRGDSP peptide in competition with O1BFS ({square}), C-S8c1 ({blacksquare}), SAT-1 ({lozenge}) and SAT-3 ({diamondsuit}). The broken line and closed circles represent the GH loop peptide (VPNLRGDLQVLA) in competition with O1BFS. Control peptides (GRGESP and VPNLRGELQVLA) had minimal effects on virus binding at 1 µM (not shown). 100% binding is the amount of virus bound in the absence of peptide. Background was measured by omitting virus from duplicate wells and was subtracted from the experimental data before conversion to percentage of control. Integrin-bound virus was detected by using an anti-serotype-specific polyclonal serum and each data point is the mean of duplicate wells. The experiment was performed three times each giving similar results (see Table 1).

 

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Table 1. IC50 values for RGD peptide competition of virus binding to {alpha}5{beta}1

 
Influence of the residue that flanks the RGD motif on binding to integrins {alpha}5{beta}1 and {alpha}v{beta}3
The viruses used in the above study were selected to represent the sequence and length heterogeneity of the GH loop of VP1 (Table 1). Despite these differences, all of the viruses displayed similar binding characteristics except for the SAT-2 virus, which consistently gave the lowest absorbance reading in the ELISA (see Figs 1 and 2). The SAT-2 virus has an arginine residue immediately following the RGD motif, whereas the other viruses used in these studies have either a leucine or methionine at this position (Table 1). We therefore tested whether changing the amino acid at the residue immediately following the RGD influenced the ability of FMDV to bind the integrins {alpha}5{beta}1 and {alpha}v{beta}3. For this study, we used two related viruses, O1K-car2 and O1K-f480. These viruses have an identical capsid sequence except for a single residue change immediately following the RGD motif. In O1K-car2, this residue is leucine, whereas in O1K-f480, it is arginine (Crowther et al., 1993 ). The GH loop of VP1 is a major antigenic site on the capsid (antigenic site-1; Xie et al., 1987 ), and the introduction of an arginine after the RGD motif results in the loss of binding by antibodies that normally recognize this site. To ensure equal detection efficiencies in the ELISA, we used a monoclonal antibody, C9, to detect integrin-bound virus, as its epitope (antigenic site-2) is common to both O1K viruses (Xie et al., 1987 ). As a consequence, C9 binds the two viruses equally well in ELISA, where the viruses are either immobilized directly onto the plate or trapped by a rabbit anti-O antibody and can be used to quantify directly the amount of virus bound to the integrin (Crowther et al., 1993 ; and data not shown).

The data shown in Fig. 5 show that both the RGDR (O1K-f480) and the RGDL (O1K-car2) viruses serve as ligands for {alpha}v{beta}3 and that the RGDR virus is a better ligand than the RGDL virus. In contrast, the opposite was found for {alpha}5{beta}1, i.e. the RGDL virus is a better ligand than the RGDR virus. Furthermore, the data in Fig. 5 show that the RGDR virus is a poor ligand for {alpha}5{beta}1, as relatively little virus binding was detected at 20 µg/ml. However, a direct comparison between the ability of the RGDL virus to bind the different integrin species ({alpha}5{beta}1 and {alpha}v{beta}3) should be made with caution, as one cannot meaningfully compare affinities of a multivalent virus in the liquid phase in independent ELISAs with different immobilized receptors.



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Fig. 5. Influence of the residue immediately following the RGD motif on virus binding to integrins {alpha}5{beta}1 and {alpha}v{beta}3. Viruses [O1K-car2 (RGDL), open bars; O1K-f480 (RGDR), hatched bars] were bound to immobilized integrins {alpha}5{beta}1 and {alpha}v{beta}3 (as indicated) at the concentrations indicated. Integrin-bound virus was detected by using a mouse monoclonal antibody (C9) that recognizes an epitope that is common to both viruses. Non-specific binding was determined by binding virus in the presence of 2 µM GRGDSP peptide and is indicated by (+PEP). Each data point is the mean of triplicate wells. Note that different virus concentrations were used for the different integrins. The experiment was performed three times, each giving similar results.

 
Identity of the residue flanking the RGD motif in outbreak strains of FMDV
In view of the importance of the amino acid at the RGD+1 position in determining integrin specificity, we determined whether the GH loop sequences in Table 1 were representative of those virus strains isolated from the field or whether they had acquired mutations during adaptation to the integrins expressed on tissue culture cell lines (Rieder et al., 1994 ). We therefore sequenced the GH loop of VP1 directly of several outbreak strains of FMDV from epithelial tissues obtained from infected animals. These sequences are shown in Table 2. All of the type O and type A viruses sequenced had a leucine residue immediately following the RGD motif, showing that this sequence predominates in naturally occurring field isolates of these serotypes. The two Asia-1 viruses sequenced had either leucine or methionine at this position. Interestingly, all of the SAT-2 viruses sequenced had an arginine at this position.


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Table 2. XRGDX motifs of FMDV sequences of RT–PCR amplicons obtained directly from epithelial suspensions

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
FMDV utilizes two major families of cell-surface receptors, RGD-dependent integrins and HSPG. Field isolates of FMDV use RGD-dependent integrins as cellular receptors in cultured cell lines. To date, {alpha}v{beta}3 is the only member of the integrin superfamily that has been shown to act as a receptor for FMDV (Berinstein et al., 1995 ), but this integrin has been reported to have limited expression on epithelial cells and cells of lymphoid origin (Springer, 1990 ; Mette et al., 1993 ; Damjanovich et al., 1992 ) and it is these cell types in which FMDV is likely to reside during the initial phase of infection of the pharynx. However, many other RGD-dependent integrins, including the Fn receptor, {alpha}5{beta}1, are expressed on these cell types, where they may have the potential to act as cellular receptors for FMDV (Springer, 1990 ; Mette et al., 1993 ; Damjanovich et al., 1992 ). The primary objective of this study was to determine whether, in the absence of HSPG and other integrins, FMDV could function as a ligand for the integrin {alpha}5{beta}1. Our results demonstrate that FMDV is an authentic ligand for {alpha}5{beta}1 in that binding is dependent on divalent cations and is mediated through an RGD sequence. An important finding of this study is that the ability of FMDV to bind {alpha}5{beta}1 is critically dependent on the activation state of the integrin. The normal physiological control mechanisms that regulate the activation state, and hence the ligand-binding potential of integrins in vivo, are at present unclear. The integrin {alpha}5{beta}1 has yet to be shown to mediate infection and at present its role as a cellular receptor for FMDV remains speculative. Nevertheless, our data show that, if {alpha}5{beta}1 functions as a receptor in the infected animal, the mechanisms that regulate the ligand-binding affinity of integrins would be expected to play an important role in infection by FMDV.

Recently, the {alpha}5{beta}1 heterodimer has been shown to have heparan sulphate chains linked covalently to both subunits (Veiga et al., 1997 ), which could potentially mediate binding of heparin-binding strains of FMDV. Of the viruses used in this study, FMDV C-S8c1 (Baranowski et al., 1998 ) and SAT-3 Zim 4/81 (unpublished observations) do not appear to bind heparin, whereas O1BFS (Jackson et al., 1996 ) and SAT-1 Bot-1/68 (unpublished observations) are heparin-binding strains. Despite the different abilities of these viruses to bind heparin, we found that an RGD-containing peptide specifically inhibited binding of all four viruses to {alpha}5{beta}1 to the same extent and potency (Fig. 4 and Table 1), implying that the interaction between FMDV and {alpha}5{beta}1 is mediated principally through the RGD-binding site on the integrin and not heparan sulphate.

Many of the capsid sequence of the viruses used in this study differ considerably, including the regions flanking the RGD motif (Table 1). Despite these differences, we noted that viruses with either a leucine or a methionine immediately flanking the RGD motif were good ligands for {alpha}5{beta}1 and displayed similar binding affinities, whereas the SAT-2 virus, which has an arginine at this position, consistently gave low absorbance values in the ELISA. These data contrast with our previous observation, made with the same FMDV strains as in this study, that the SAT-2 (RGDR) and SAT-3 (RGDM) viruses are better suited to binding {alpha}v{beta}3 than viruses of the other serotypes, which have an RGDL motif (Jackson et al., 1997 ). A longer RGD-containing peptide, with its sequence derived from the GH loop of VP1 of O1BFS, was found to be a more potent inhibitor of O1BFS binding to {alpha}5{beta}1 (IC50 77 nM) than reported previously for O1BFS binding to {alpha}v{beta}3 (IC50 1 µM; Jackson et al., 1997 ). The above observations are consistent with the notion that the identity of the amino acid immediately following the RGD motif could have a major influence on the ability of the different FMDV strains to bind the individual integrin species. Two previous studies with FMDV have implicated the residues following the RGD, including that at the RGD+1 position, in receptor recognition (Rieder et al., 1994 ; Mateu et al., 1996 ). We therefore compared the binding of two closely related strains of type O FMDV, which differ only in the arginine to leucine change at the residue immediately following the RGD motif, to {alpha}5{beta}1 and {alpha}v{beta}3. Our data show that both viruses (RGDL and RGDR) serve as ligands for {alpha}v{beta}3, although the virus with an arginine residue immediately following the RGD motif was found to be a better ligand than the virus with a leucine at this position. In contrast, the opposite was found to be the case for {alpha}5{beta}1, in that the RGDL virus is a better ligand than the RGDR virus and, furthermore, the RGDR virus is a poor ligand for this integrin. From these data, we conclude that viruses that have a leucine immediately following the RGD motif can serve as ligands for both {alpha}5{beta}1 and {alpha}v{beta}3, whereas viruses that have an arginine at this position become better ligands for {alpha}v{beta}3, but at the expense of becoming poor ligands for {alpha}5{beta}1.

Our data with FMDV are consistent with published evidence of sequence preferences of {alpha}5{beta}1 and {alpha}v{beta}3, obtained by using random phage-display libraries (Koivunen et al., 1993 ; Healy et al., 1995 ). These experiments have shown that both {alpha}v{beta}3 and {alpha}5{beta}1 have a strong preference for peptides containing an RGD motif, but that {alpha}v{beta}3 is tolerant of several different amino acids immediately following the RGD, including basic residues such as arginine and lysine, consistent with its role as a multifunctional receptor that binds a broad range of ligands. In contrast, {alpha}5{beta}1 binds a more restricted set of peptides, where the RGD is often followed by a hydrophobic residue, preferably leucine (Koivunen et al., 1993 ).

Across six of the FMDV serotypes, the majority of viruses have a leucine immediately following the VP1 GH loop RGD motif; the exception is the SAT-2 viruses, where this residue is most commonly arginine (N. J. Knowles and A. R. Samuel, unpublished data). Propagation of virulent, wild-type FMDV in cultured cell lines has been shown to result in the selection of variant viruses with different residues flanking the RGD motif (Rieder et al., 1994 ). All of the viruses used in this study have been passaged in BHK cells and it is possible that the predominance of leucine immediately following the RGD could have resulted from such a selection process. However, the sequence data in Table 2, obtained directly from infected animals, show that leucine predominates after the RGD in outbreak strains, at least in the O and A serotypes. The fact that a leucine residue is selected at the RGD+1 position in an infected animal may enable infection to be mediated by either {alpha}v{beta}3 or {alpha}5{beta}1, thereby extending the range of integrin receptors used in vivo.

Several other picornaviruses have RGD sequences that are most commonly followed by a leucine residue, which has led to suggestions that these viruses may use the same RGD-dependent integrins as receptors (Roivainen et al., 1994 ; Chang et al., 1992 ; Nelsen-Salz et al., 1999 ; Zimmermann et al., 1996 ; Jung et al., 1998 ; Hyypiä et al., 1992 ; Ghazi et al., 1998 ; Oberste et al., 1998 ). It is interesting to speculate that many viruses, like FMDV, maintain a leucine residue immediately after the RGD motif to permit the use of several different RGD-binding integrins as receptors in vivo.


   Acknowledgments
 
We thank M. Pitkeathly and S. Shah for the peptides and Sue Craig and David Ansell for their technical assistance. This work was supported by MAFF.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 16 November 1999; accepted 2 February 2000.