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
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Abstract |
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Introduction |
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Integrins are a family of transmembrane /
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
; OToole 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,
v
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
v
1,
v
5,
v
6 and
5
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
v
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
5
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
-galactosidase or on the surface of hepatitis B virus core particles, functions as a ligand for
5
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
1 integrins that had been enriched for
5
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
5
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
5
1 and
v
3. The data are discussed in relation to the GH loop sequence of naturally occurring field isolates of FMDV.
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Methods |
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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-
1 antibody 9EG7 (Bazzoni et al., 1995
) and the inhibitory antibodies P4C10 (anti-
1, BRL) and JBS5 (anti-
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).
Solid-phase binding assay.
The standard assay was carried out as follows. Plastic 96-well plates were coated with integrin 5
1 (~1 µg/ml) in coating buffer (25 mM TrisHCl, 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 TrisHCl, pH 7·4, 150 mM NaCl, 1 mM MnCl2 3% radioimmunoassay grade BSA (ICN)]. Virus, in binding buffer, was added to the wells for 23 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 TBS3% BSA containing the appropriate cation.
Integrin v
3 (Chemicon) was coated at 0·5 µg/ml in coating buffer (25 mM TrisHCl, 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 TrisHCl, 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
5
1.
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 phenolchloroform 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 manufacturers 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.
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Results |
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The data shown in Fig. 5 show that both the RGDR (O1K-f480) and the RGDL (O1K-car2) viruses serve as ligands for
v
3 and that the RGDR virus is a better ligand than the RGDL virus. In contrast, the opposite was found for
5
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
5
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 (
5
1 and
v
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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Discussion |
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Recently, the 5
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
5
1 to the same extent and potency (Fig. 4
and Table 1
), implying that the interaction between FMDV and
5
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
5
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
v
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
5
1 (IC50 77 nM) than reported previously for O1BFS binding to
v
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
5
1 and
v
3. Our data show that both viruses (RGDL and RGDR) serve as ligands for
v
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
5
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
5
1 and
v
3, whereas viruses that have an arginine at this position become better ligands for
v
3, but at the expense of becoming poor ligands for
5
1.
Our data with FMDV are consistent with published evidence of sequence preferences of 5
1 and
v
3, obtained by using random phage-display libraries (Koivunen et al., 1993
; Healy et al., 1995
). These experiments have shown that both
v
3 and
5
1 have a strong preference for peptides containing an RGD motif, but that
v
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,
5
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
v
3 or
5
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.
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Acknowledgments |
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References |
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Received 16 November 1999;
accepted 2 February 2000.