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Journal of Virology, May 2004, p. 4533-4540, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4533-4540.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Integrin vß8 Functions as a Receptor for Foot-and-Mouth Disease Virus: Role of the ß-Chain Cytodomain in Integrin-Mediated Infection

Terry Jackson,^1* Stuart Clark,¹ Stephen Berryman,¹ Alison Burman,¹ Stephanie Cambier,² Dezhi Mu,² Stephen Nishimura,² and Andrew M. Q. King¹

Department of Molecular Biology, Institute for Animal Health, Pirbright, Surrey GU24 ONF, United Kingdom,¹ Department of Pathology, University of California at San Francisco, San Francisco, California 94143²

Received 22 September 2003/ Accepted 5 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three v integrins, vß1, vß3, and vß6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human ß8 cDNA and expression of vß8 at the cell surface. The involvement of vß8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the vß8 heterodimer or the v chain. Similar results were obtained with a chimeric vß8 including the ß6 cytodomain (vß8/6), showing that the ß6 cytodomain can substitute efficiently for the corresponding region of ß8. In contrast, virus binding to vß6 including the ß8 cytodomain (vß6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the vß6 chimera was recognized poorly by antibodies specific for the ectodomain of vß6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the ß6 cytodomain is important for maintaining vß6 in a conformation required for productive infection by FMDV.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease, a severe vesicular disease of cloven-hoofed animals including domesticated ruminants and pigs. The virus exists as seven serotypes, which are members of the genus Aphthovirus of the family Picornaviridae (35). The virion consists of an 8.5-kb strand of RNA enclosed within an icosahedral capsid formed from 60 copies each of four proteins, VP1 to VP4 (1).

Two classes of cell surface receptors that mediate FMDV infection have been identified (30). Theses are the integrins (7, 31, 33) and heparan sulfate (HS) proteoglycans (HSPGs) (29). The ability to use HSPGs as receptors appears to be restricted to strains of FMDV that have been multiply passaged through cultured cell lines (4, 5, 22, 41, 52, 58), and presently there is no convincing evidence of a role for HS in cell entry by field viruses. Instead, field viruses are dependent on integrin receptors to initiate infection in vitro, and integrins are believed to be the receptors used in the infected animal. Recently, two independent studies have shown that certain strains of FMDV can infect cultured cells via an entry pathway that is independent of both integrins and cellular HS, implying the existence of a third, as yet unidentified receptor family (4, 65).

Integrins are a family of integral membrane receptors with distinct ligand-binding specificities and tissue distributions. They contribute to a variety of cellular functions, including cell-cell and cell-matrix adhesion, and exist in alternative low- and high-affinity states, enabling them to transmit signals both into and out of cells (19, 25). Each receptor molecule is a heterodimer of two type 1 transmembrane subunits, and ß, each of which has a large extracellular domain and in most cases a short cytoplasmic tail. Most members of the integrin family recognize their ligands by binding to short linear peptide sequences, and several, including vß1, vß3, vß5, vß6, vß8, 5ß1, and 8ß1, recognize the arginine-glycine-aspartic acid (RGD) motif. To date, three RGD-dependent integrins, vß1, vß3, and vß6, have been reported to function as receptors for FMDV (7, 31, 33). Virus attachment to the integrin is mediated through a highly conserved RGD tripeptide, located at the apex of a long surface loop, the GH loop of VP1 (6, 24, 31, 32, 33, 38, 39, 42, 44, 56, 59). However, despite having an RGD, FMDV appears unable to use any of the RGD-dependent integrins as receptors to initiate infection, and evidence for vß5 and 5ß1 as receptors has been consistently negative (4, 21, 33, 43, 52).

The integrin vß6 is of particular interest because, in our experience, it is a much more active receptor for FMDV than either vß1 or vß3 and is expressed exclusively in epithelial cells, which are the preferred cell type infected by FMDV in vivo. It is also unusual among integrins in binding only a small number of ligands, including the latency-associated protein (LAP) component of transforming growth factor ß1 (TGF-ß1) and TGF-ß3 (27, 40, 50, 57, 62, 64). The amino acid sequences that immediately follow the RGD of LAP-1 (RGDLATI), LAP-3 (RGDLGRL), and FMDV (RGDLQVL) are similar to each other, which suggests that these ligands may share common integrin receptors. Recently, LAP-1 has been shown to be a ligand for vß8 also, and this prompted us to investigate whether vß8 could serve as a receptor for FMDV (49). In this report, we show that SW480 cells become susceptible to FMDV following transfection with the integrin ß8 subunit and stable expression of vß8 at the cell surface. The involvement of vß8 in infection was confirmed in competition experiments showing that virus binding and infection are inhibited by function-blocking monoclonal antibodies (MAbs) specific for the vß8 heterodimer or the v chain.

The cytodomains of the and ß chains are critically important for many of the functional properties of integrins, including the regulation of ligand-binding affinity, linkage to the cytoskeleton, formation of signaling complexes, and integrin-mediated uptake of ligands (8, 19, 28, 55, 60, 63). As a first step toward understanding the events that follow the attachment of a virus to its integrin receptor, we have studied the role of the ß6 cytodomain in vß6-mediated infection (47). These studies showed that although the ß6 cytodomain was not required for virus binding to vß6, the integrity of this domain was essential for vß6-mediated infection (47). In the present study, we have investigated further the role of the ß-chain cytodomain by using chimeric vß6 and vß8 integrins in which the cytodomains of the ß chains have been exchanged (vß6/8 and vß8/6, respectively). These studies show that the ß6 cytodomain can substitute efficiently for the corresponding domain of ß8. In contrast, although FMDV bound to cells expressing the vß6/8 chimera, and did so through an RGD-dependent interaction, this binding led only to very inefficient infection. Furthermore, the vß6/8 chimera was recognized poorly by antibodies specific for the ectodomain of vß6 and displayed an altered sequence-binding specificity for RGD-containing peptides. Together, these data suggest that the ß6 cytodomain is important for maintaining vß6 in a conformation required for productive infection by FMDV.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and viruses. BHK cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (FCS), 20 mM glutamine, penicillin (100 SI units/ml), and streptomycin (100 µg/ml). Mock- and integrin transfected SW480 cells expressing either wild-type vß6, wild-type vß8, or the chimeric ß subunits were cultivated in Dulbecco's modified Eagle medium supplemented with 10% FCS, 20 mM glutamine, penicillin (100 SI units/ml), streptomycin (100 µg/ml), and 4 µg of puromycin (Sigma)/ml. Construction of cells expressing wild-type vß6, wild-type vß8, and the vß6/8 chimera has been described previously (15, 49). The ß8/6 chimera was made by splice-overlap PCR mutagenesis using the mutagenic primers 5'-CTGATCATTAGACAGGTGATACTACAATGGAAGCTACTGGTGTCATTTCAT-3' and 5'-ATGAAATGACACCAGTAGCTTCCATTGTAGTATCACCTGTCTAATGATCAG-3', joining W711 to K731 of the respective ß8 and ß6 open reading frames. Transduction and selection were performed as described previously (15). Construction and cultivation of cells expressing deletions in the ß6 cytodomain (SW480-T1, -T3, and -T5) have been described previously (2, 16, 50). The virus used in this study was FMDV strain O1Kcad2. This virus does not bind HS and is dependent on integrins as its sole receptor family. For infectivity assays, virus stocks were prepared by using primary bovine thyroid cells. In all assays, the multiplicity of infection (MOI) was based on the virus titer on BHK cells. Virus purification on sucrose gradients was performed as described previously (17).

Antibodies and peptides. The FMDV RGD peptide, with its sequence derived from the GH loop of VP1 of type O virus (FMDV-RGD; VPNLRGDLQVLA), and the control RGE version were synthesized in the peptide synthesis facility at the Oxford Centre for Molecular Science, New Chemistry Laboratory, Oxford, United Kingdom. The GRGDSP and GRGESP peptides were purchased from Novabiochem. Anti-integrin antibodies used in these studies were10D5 (mouse immunoglobulin G2a [IgG2a]) and E7P6 (mouse IgG1) against vß6, R6G9 (mouse IgG2a) against ß6, P1F6 (mouse IgG1) against vß5, and 6S6 (mouse IgG1) against ß1 (all from Chemicon) and SAM-1 (mouse IgG2b) against 5ß1 (Serotec). Other MAbs used were 14E5 (IgG1) and 37E5 (IgG2a) against vß8 and the anti-v MAb L230 (mouse IgG1). The anti-FMDV MAbs B2 (mouse IgG1) and D9 (mouse IgG2a), which recognize antigenic site 1 of type O FMDV (45), were purified by using protein A (Pierce) according to the manufacturer's instructions.

Infectious center assay. Cells were harvested by using EDTA and were washed in cell culture medium. One million cells were collected by centrifugation, resuspended in 100 µl of Tris-buffered saline (pH 7.4) containing 1 mM CaCl₂ and 0.5 mM MgCl₂, and infected with FMDV O1Kcad2 (MOI, 0.3) at 37°C for 1 h with continuous rotation. Following infection, virus that remained on the outsides of the cells was inactivated by addition of 1 ml of 0.1 M citric acid buffer (pH 5.2) for 2 min. The cells were washed with phosphate-buffered saline (PBS), pH 7.5, containing 2 mM CaCl₂ and 1 mM MgCl₂ and then resuspended in 300 µl of the same buffer supplemented with 0.5% FCS. Dilutions of the infected cells (100 µl) were layered onto subconfluent monolayers of BHK cells as described previously (33). The monolayers were incubated at 37°C for 40 to 48 h, after which the infectious centers were visualized as plaques by staining with methylene blue-4% formaldehyde in PBS (pH 7.5). In the competition experiments, anti-integrin antibodies and peptides (0.1 mM) were added to the cells for 0.5 h at room temperature prior to the addition of virus, and infection was initiated by incubation at 37°C for 45 min. Following infection, virus that remained on the outsides of the cells was acid inactivated, and the cells were plated onto BHK monolayers as described above.

Flow cytometry analysis. (i) Integrin expression. Cells were harvested by using EDTA and were resuspended at 5 x 10⁶ per ml in a solution containing Tris-buffered saline (pH 7.5), 1 mM CaCl₂, 0.5 mM MgCl₂, 2% goat serum, and 3% bovine serum albumin (buffer A). Cells (30 µl) were collected by centrifugation and incubated with primary antibodies (10 µg/ml in buffer A) on ice for 0.5 h. The cells were then washed with buffer A and incubated on ice for 25 min with secondary antibodies conjugated with R-phycoerythrin (Southern Biotechnology Associates). The cells were then washed twice with buffer A and resuspended in PBS (pH 7.5)-2 mM CaCl₂-1 mM MgCl₂ containing 1% paraformaldehyde. Fluorescent staining was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson) and by counting 10,000 cells per sample. Background fluorescence was determined by omitting the primary antibody from the assay.

(ii) Virus binding assay. Cells were prepared in buffer A as described above and then incubated with O1Kcad2 (10 µg/ml) for 0.5 h on ice. The cells were then washed with buffer A and incubated with the anti-type O MAb B2 (10 µg/ml), followed by an R-phycoerythrin-conjugated goat anti-mouse IgG1 antibody. Background fluorescence was determined by omitting either the virus or MAb B2 from the assay. These two control conditions gave nearly identical results.

(iii) Competition experiments. Competing MAbs and peptides were added to the cells for 0.5 h on ice before the addition of virus (10 µg/ml) for a further 0.5 h. The cells were then washed with buffer A, and cell-bound virus was detected by using an anti-type O FMDV MAb. When 10D5, 37E5, or SAM-1 was used as a competitor, virus was detected by using MAb B2. When P1F6 was used as a competitor, virus was detected by using MAb D9. Anti-FMDV antibodies were detected by using R-phycoerythrin-conjugated goat anti-mouse IgG isotype-specific antibodies. For these experiments, additional controls were performed to verify that the R-phycoerythrin-conjugated isotype-specific antibodies were not cross-reactive for the competing MAb.

Top Abstract Introduction Materials and Methods Results Discussion References

 
To determine whether integrin vß8 could function as a receptor for FMDV, we compared SW480 cells that had been transfected to stably express vß8 with cells transfected with the expression plasmid alone (mock transfected). To further understand the role of the ß-chain cytodomain in integrin-mediated infection, we included in these studies cells expressing either wild-type vß6 or chimeric vß8 or vß6 in which the cytodomains of the ß chains had been exchanged (SW480vß8/6 and SW480vß6/8, respectively).

Initially, we used flow cytometry to confirm the integrin expression profiles on transfected cells. SW480 cells normally express vß5 and 5ß1 as their only RGD-binding integrins (Fig. 1) (62). However, upon transfection with integrin ß-chain cDNA, they are capable of expressing "new" vß combinations as functional heterodimers. Figure 1 shows that expression of vß5 was lower on cells transfected with ß-chain cDNA than on mock-transfected cells, presumably as a result of competition between the endogenous and transfected ß chains for the v subunit, whereas expression of 5ß1 was not altered. Cells transfected with either the wild-type ß8 chain or the ß8/6 chimera were found to express similar amounts of vß8 on the cell surface. Similarly, by use of an antibody specific for the ectodomain of the ß6 chain, cells transfected with either wild-type ß6 or the ß6/8 chimera were found to express similar amounts of ß6 (Fig. 1). Because ß6 is expressed at the cell surface only as a heterodimer with the v chain, this observation indicates that the transfected cells express similar amounts of vß6 or vß6/8, respectively. However, despite the fact that ß6 is expressed at similar levels on transfected cells, antibodies specific for the vß6 heterodimer (MAbs 10D5 and E7P6) appeared to recognize the vß6/8 chimera less efficiently than they recognized wild-type vß6 (Fig. 1). This reduction in expression of the epitopes for MAbs 10D5 and E7P6 suggests that inclusion of the ß8 cytodomain maintains vß6 in a conformation which is recognized poorly by MAbs specific for the ectodomain of vß6. During the course of the experiments reported here, levels of integrin expression on transfected cells were determined by flow cytometry and did not change significantly from those shown in Fig. 1.

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FIG. 1. Flow cytometric analysis of RGD-dependent integrins expressed on mock- and ß-transfected SW480 cells. Mock-transfected cells (SW480 mock) and cells transfected with wild-type ß6 (SW480 vß6), wild-type ß8 (SW480 vß8), or the chimeric ß subunit ß6/8 (SW480 vß6-8) or ß8/6 (SW480 vß8-6) were incubated with (open histogram) or without (solid histogram) the indicated anti-integrin antibody, followed by an R-phycoerythrin-conjugated goat anti-mouse isotype-specific secondary antibody. The mean fluorescence intensity is shown for each antibody.

Next, we compared infection of the transfected cells by using an infectious center assay, which permits the number of productive infectious events to be quantified. Table 1 shows that cells expressing vß8 are more susceptible to infection by FMDV than mock-transfected cells. Similar numbers of infectious centers were obtained with cells expressing either wild-type vß8 or the vß8/6 chimera; certainly there was no evidence that the domain substitution reduced the receptor activity of the integrin. As expected, expression of vß6 also resulted in increased susceptibility to infection relative to that of mock-transfected cells. Only a small number of infectious centers were obtained for cells transfected with the ß6/8 chimera.

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TABLE 1. Infection of transfected cellsa

To confirm the role of vß8 in infection, we performed competition experiments using function-blocking MAbs specific for either the vß8 heterodimer (MAb 37E1) or the v chain (MAb L230). Figure 2 shows that preincubation of vß8-expressing cells with these antibodies inhibited infection, whereas MAbs to vß5 or the ß1 chain did not have a significant inhibitory effect. A combination of the MAbs to vß8 and vß5 (10 µg/ml each) did not result in a greater inhibitory effect than that obtained with the vß8 MAb alone (data not shown). Similarly, preincubation of vß8-expressing cells with an RGD-containing peptide whose sequence is derived from the FMDV RGD site (FMDV-RGD) (see Materials and Methods) inhibited infection by more than 90% (data not shown). These data show that vß8 functions as a receptor for FMDV and that its ability to mediate infection is not diminished by replacing the ß-chain cytodomain with the corresponding region of ß6. In contrast, the ability of vß6 to mediate FMDV infection was almost completely inhibited (97%) by substitution of the ß8 cytodomain.

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FIG. 2. Infection of ß8-transfected SW480 cells is specifically inhibited by MAbs to the v subunit and the vß8 heterodimer. ß8-transfected cells (SW480vß8) were incubated with antibodies against vß5 (P1F6), vß8 (37E1), ß1 (6S6), or v (L230) prior to infection by O1Kcad2 at an MOI of 0.3 PFU/cell, and the infected cells were used in an infectious center assay. Numbers of infectious centers are expressed as percentages of the number obtained in the absence of competing antibodies, taken as 100%. Data are means (± standard errors of the means) from three independent experiments, each carried out in duplicate.

The data described above show that vß8 promotes infection by FMDV. To gain a better understanding of the role of vß8 in infection, we determined the abilities of the transfected cells to bind FMDV. Figure 3 shows that the increased susceptibility to infection of cells expressing vß8 is correlated with an increase in virus binding. Similar levels of virus binding were obtained with cells expressing either wild-type vß8 or the vß8/6 chimera, consistent with the similar levels of integrin expressed by these cells (Fig. 1). Figure 3 also shows that, in agreement with previous observations (33), FMDV binds to SW480 cells expressing wild-type vß6. Interestingly, the very poor susceptibility to infection of cells expressing the vß6/8 chimera was not fully reflected in the binding data, which show almost one-quarter of the virus binding remaining relative to that of cells expressing wild-type vß6.

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FIG. 3. Flow cytometric analysis of FMDV binding to mock- and ß-transfected SW480 cells. Histograms show FMDV O1Kcad2 binding to mock-transfected (SW480-mock) and cells expressing vß6 (SW480/ß6), vß8 (SW480/ß8), vß6/8 (SW480/ß6-8), or vß8/6 (SW480/ß8-6). Virus binding (open histograms) was detected by using the anti-FMDV antibody B2 followed by an R-phycoerythrin-conjugated goat anti-mouse IgG1 secondary antibody. Mean fluorescence intensities are given. Solid histograms, background fluorescence (see Materials and Methods).

To confirm the direct involvement of vß8 as a virus attachment receptor, we carried out competition experiments using the anti-integrin MAbs described above. Figure 4 shows that MAbs to vß8 (37E5) or the v chain (L230) inhibited virus binding to cells expressing wild-type vß8, whereas MAbs to vß5 or 5ß1 did not have an inhibitory effect. Similar observations were made for cells expressing the vß8/6 chimera (data not shown), confirming that inclusion of the ß6 cytodomain does not interfere with virus binding to vß8. In agreement with a previous observation (33), the anti-vß6 MAb, MAb 10D5, inhibited FMDV binding to cells expressing wild-type vß6 (data not shown). We were unable to reproduce these observations with cells expressing the vß6/8 chimera, because MAb 10D5 is the only currently available function-blocking MAb to vß6, and this MAb has a low affinity for these cells (Fig. 1). However, in view of the fact that virus binding was inhibited preferentially by the FMDV peptide (see below and Fig. 5), a characteristic of vß6, it is likely that the chimeric vß6/8 integrin is a receptor for FMDV attachment.

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FIG. 4. Anti-integrin antibodies inhibit the binding of FMDV to ß8-transfected SW480 cells. ß8-transfected cells (SW480vß8) were incubated with antibodies to vß5 (P1F6), vß8 (37E1), 5ß1 (SAM-1), or v (L230) at 6 µg/ml prior to the addition of virus (O1Kcad2; 10 µg/ml), and the cells were analyzed for bound virus by flow cytometry (see Materials and Methods). Data are expressed as percentages of the level of binding in the absence of the competing antibody (set at 100%) and are means of two independent experiments, each carried out using triplicate samples, that gave near-identical results.

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FIG. 5. Binding of FMDV to ß-transfected SW480 cells is inhibited by RGD peptides. ß-transfected cells expressing wild-type vß8 (SW480/vß8) (A), wild-type vß6 (SW480/vß6) (B), or chimeric vß6/8 (SW480/vß6-8) (C) were incubated with an RGD-containing peptide (VPNLRGDLQVLA [solid bars] or GRGDSP [open bars]) or the control RGE version (VPNLRGELQVLA [hatched bars] or GRGESP [checked bars]) prior to the addition of FMDV O1Kcad2 (10 µg/ml). Cell-bound virus was detected by flow cytometry using the anti-FMDV antibody B2 followed by an R-phycoerythrin-conjugated goat anti-mouse IgG1 antibody. Data are means from two independent experiments, each carried out by using triplicate samples, that gave nearly identical results.

The anti-vß8 MAb (MAb 37E1) was found to inhibit virus binding to cells expressing vß8 by 60% (Fig. 4) despite inhibiting infection by 85% (Fig. 2). Increasing the concentration of this MAb to 25 µg/ml did not inhibit virus binding beyond that shown in Fig. 4. Similar observations were made with cells expressing the vß8/6 chimera (data not shown). At present we do not know why the anti-vß8 MAb did not inhibit virus binding to cells expressing vß8 more efficiently. However, when used at a high concentration, MAb 37E1 (IgG2a) could be detected by the anti-IgG1 conjugated antibody used to detect virus binding (see Materials and Methods). This cross-reactivity could, in part, account for the apparent residual virus binding in the presence of MAb 37E1. Alternatively, since the anti-v MAb inhibited virus binding to a greater extent that the anti-vß8 MAb, it is possible that more than one v integrin may serve as a receptor for virus attachment to the transfected cells. However, this explanation is unlikely for two reasons. First, SW480 cells normally express only one v integrin, vß5, and this integrin is not a receptor for FMDV on these cells (Table 1; Fig. 3). Second, an antibody to vß5 did not inhibit virus binding or infection of cells expressing wild-type vß8 (Fig. 4), again suggesting that vß5 is not involved in virus attachment.

Ligand binding to integrins is differentially regulated by divalent cations, and manganese (Mn²⁺) ions are known to enhance ligand binding to several integrin receptors (34, 36, 37, 48). It has been shown previously that Mn²⁺ ions enhance FMDV binding to vß1 and vß3 (31, 32), whereas this cation does not affect FMDV binding to vß6 (33). FMDV binding to cells expressing wild-type vß8 in the presence of 1 mM MnCl₂ was not increased above that obtained in the presence of calcium and magnesium alone (data not shown).

The binding of FMDV to its integrin receptors is RGD dependent and is inhibited by synthetic peptides containing this motif; moreover, viruses with mutations at the RGD site fail to bind to cells (see the introduction). Previously it has been shown that the binding of FMDV to its integrin receptors is differentially sensitive to RGD-containing peptides. Thus, whereas an FMDV RGD peptide (VPNLRGDLQVLA) inhibits virus binding to vß1, vß3, and vß6, a shorter RGD-containing peptide (GRGDSP) is effective only for vß1 and vß3 and does not inhibit virus binding to vß6 (31, 32, 33). To determine whether FMDV binding to vß8 is also differentially sensitive to RGD-containing peptides, we used the two peptides described above in competition experiments to inhibit virus binding to vß8. Figure 5A shows that FMDV binding to vß8 was inhibited by either of these RGD-containing peptides in a sequence-specific manner, since the control RGE versions of these peptides had only a minimal effect on virus binding at the highest concentration used. The FMDV peptide was found to be a more potent inhibitor of virus binding to vß8 than the GRGDSP peptide, suggesting that high-affinity binding of the FMDV peptide to vß8 may also be dependent on residues that lie outside of the RGD motif.

To understand further the nature of the virus binding to the vß6/8 chimera, we also performed peptide competition experiments using cells expressing this integrin (Fig. 5C). As expected, virus binding to cells expressing wild-type vß6 was inhibited by the FMDV peptide but not by the GRGDSP peptide (Fig. 5B), confirming previous observations (31). Figure 5C shows that virus binding to cells expressing vß6/8 is also inhibited by the FMDV peptide; however, much less peptide was required to inhibit virus binding to these cells than to cells expressing wild-type vß6. It is worth recalling that the domain substitution in vß6/8 was also associated with a reduced ability to bind virus (Fig. 3), and it seems likely that both properties may reflect a reduced affinity of the vß6/8 chimera for FMDV relative to that of wild-type vß6. In addition, the vß6/8 chimera appeared to display a relaxed sequence-binding specificity relative to that of wild-type vß6, since the GRGDSP peptide was found to inhibit virus binding to cells expressing this chimera (Fig. 5C).

It has been reported previously that deletions in the ß6 cytodomain do not interfere with FMDV binding to vß6, whereas the same deletions reduce the ability of this integrin to mediate infection (47). Specifically, truncation of the 17 C-terminal residues of the ß6 chain (SW480-T3) does not significantly reduce the ability of vß6 to mediate infection, whereas infection of cells expressing receptors lacking the entire ß6 cytodomain (SW480-T1) or containing an internal deletion within this domain (SW480-T5) is greatly reduced (47). These data have suggested that the ß6 cytodomain may be required for a postattachment step(s) in FMDV infection. In the present study, we have observed that cells expressing the vß6/8 chimera show characteristics similar to those expressing the T1 and T5 deletions, i.e., they support FMDV binding, but infection is greatly reduced. Because the binding of MAbs to the ectodomain of vß6 (MAbs 10D5 and E7P6) was reduced on cells expressing the vß6/8 chimera (Fig. 1), we investigated whether the epitopes for these MAbs are expressed on cells expressing receptors with ß6 cytodomain deletions (SW480-T1, -T3, and -T5). Figure 6 shows the results of a flow cytometric analysis of vß6 expression on these cells. Binding of the anti-vß6 MAbs (MAbs 10D5 and E7P6) is shown relative to that of MAb R6G9, which recognizes the ectodomain of the ß6 chain. The binding of MAbs 10D5 and E7P6 was similar for cells expressing wild-type vß6 or the T3 deletion. In contrast, like that for cells expressing the vß6/8 chimera, binding of MAbs 10D5 and E7P6 to cells expressing the T1 or T5 deletion was greatly reduced (Fig. 6). Thus, as with the vß6/8 chimera, the majority of vß6 expressed on the SW480-T1 and -T5 cells is maintained in a conformation that is both poorly recognized by MAbs for the vß6 heterodimer and unable to mediate infection by FMDV.

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FIG. 6. Bar graphs showing results of flow cytometric analysis of vß6 receptors containing deletions in the ß6 cytodomains. Cells expressing either wild-type vß6 (wt-B6) (SW480vß6) or vß6 containing deletion T1 (SW480-T1), T3 (SW480-T3), or T5 (SW480-T5) in the ß6 cytodomain were analyzed by flow cytometry for expression of epitopes present on the ectodomain of the ß6 chain (MAb R6G9) or the vß6 heterodimer (10D5 and E7P6). Data are ratios of the mean fluorescence intensity (MFI) obtained with the anti-vß6 MAbs to the MFI for MAb R6G9. Means ± standard deviations from three independent experiments, each carried out in triplicate, are shown.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Field strains of FMDV use integrins as receptors to initiate infection in vitro, and integrins are believed to perform the same role in the infected animal (52). Prior to this study, three integrins, vß1, vß3, and vß6, had been reported to function as receptors for FMDV. In the present study we have shown that a fourth v integrin, vß8, can also function as a receptor for FMDV. The main evidence in support of this finding is as follows: (i) SW480 cells are normally nonpermissive for FMDV but are made susceptible to infection by transfection with ß8 cDNA and expression of vß8 at the cell surface; (ii) virus attachment to the transfected cells is inhibited by function-blocking MAbs specific for either the vß8 heterodimer or the v chain; (iii) in agreement with the above observations, infection of cells expressing vß8 is also inhibited by the same antibodies.

Binding of FMDV to its integrin receptors is RGD dependent and is inhibited by synthetic peptides containing this motif (see the introduction and Fig. 5). However, the binding of FMDV to its various integrin receptors is differentially sensitive to such peptides. Specifically, whereas an FMDV-derived RGD peptide (FMDV-RGD; VPNLRGDLQVLA) inhibits virus binding to vß1, vß3, and vß6, a shorter RGD-containing peptide (GRGDSP) is effective only for vß1 and vß3, failing to inhibit virus binding to vß6 (31, 32, 33). These data suggest that residues within the FMDV peptide, in addition to RGD, may be required for high-affinity binding to vß6. In the present study, we have shown that both of these peptides inhibit FMDV binding to vß8; however, in contrast to vß1 and vß3, for which the GRDGSP peptide was found to be the more potent inhibitor, the FMDV RGD peptide is the more potent inhibitor of binding to vß8. These data suggest that, as for vß6, residues other than RGD may be required for high-affinity ligand binding to vß8.

Studies to determine the role of the integrin cytodomains in FMDV infection have obtained contrasting results. Neff and Baxt (53) have reported that deletion of the cytodomain from either the or the ß chain does not interfere with the ability of vß3 to mediate infection, whereas Miller et al. have shown that certain deletions within the ß6 cytodomain result in vß6 receptors that, although still able to bind FMDV, are no longer competent to mediate infection (47). Specifically, a deletion mutant lacking the 17 C-terminal residues of the ß6 cytodomain (the T3 deletion) binds FMDV and mediates infection similarly to the wild-type integrin (47), whereas deletion mutants either with an internal deletion in the ß6 cytodomain (the T5 deletion) or lacking this domain completely (the T1 deletion) bind virus and mediate infection inefficiently (47). These data have suggested that the ß6 cytodomain may be required for a postattachment event(s) in infection. In the present study, we have investigated further the role of the ß-chain cytodomain in integrin-mediated infection. This study has shown that replacement of the ß8 cytodomain with the corresponding region of ß6 does not affect the expression of vß8 at the cell surface or the ability of vß8 to bind virus and mediate infection. In contrast, a chimeric vß6 including the ß8 cytodomain (vß6/8) shared the characteristics of the vß6 deletion mutants (T1 and T5): it bound FMDV but was unable to mediate infection. The vß6/8 chimera is recognized poorly by antibodies specific for vß6, suggesting that the presence of the ß8 cytodomain alters the conformation of the vß6 ectodomain. This observation led us to reexamine vß6 expression on SW480 cells expressing the ß6 cytodomain deletion mutants (T1, T3, and T5). This study has shown that, as for the vß6/8 chimera, the binding of the vß6-specific MAbs is also reduced when vß6 includes the T1 or T5 modified ß chain. In contrast, these MAbs recognize similarly vß6 containing the T3 or wild-type ß6 chain.

At present we do not know why the binding of virus to cells expressing the vß6/8 chimera or the T1 or T5 deletion mutant does not lead to infection. The domain substitution in the vß6/8 chimera was associated both with a reduced ability to bind FMDV (Fig. 3) and with a reduction in the amount of RGD peptide required to inhibit this binding (Fig. 5C). In addition, the sequence-binding specificity for the vß6/8 chimera was altered from that for wild-type vß6. These properties may reflect a reduced affinity of the vß6/8 chimera for FMDV. Taken together, our data are consistent with the hypothesis that the ß6 cytodomain is required to maintain the ectodomain of vß6 in a conformation that is necessary for both high-affinity binding of FMDV and subsequent infection.

Alternatively, given the role of ß-chain cytodomains in endocytosis (60, 63), the loss of susceptibility to infection observed for cells expressing the vß6/8 chimera or the T1 or T5 deletion mutation may be due to a defect in virus internalization. However, sequences required for integrin-mediated virus internalization are missing from the ß8 cytodomain. The ß8 cytodomain is almost completely divergent in primary sequence from the other ß integrin cytodomains, which in general are highly homologous (54, 60). Thus, it is possible that vß8 mediates productive FMDV infection through a mechanism independent of its ß-chain cytodomain. This idea is supported by previous studies which demonstrate that vß8 is functionally competent as a TGF-ß-activating receptor even in the absence of its cytodomain (49).

A third possibility is that the cytodomain of the ß6 chain is required for membrane penetration, thereby permitting entry of the viral RNA genome into the cellular cytoplasm. This role has been proposed for the cytodomain of the ß5 chain in vß5-mediated infection by adenovirus (61). In the case of vß5, it is the 3 C-terminal residues of the ß5 chain that are most important for membrane penetration. However, these residues are different in the ß6 chain, and their deletion does not inhibit vß6-mediated infection by FMDV (47). Therefore, it would appear that, if the ß6 cytodomain is needed for virus penetration into the cell, it works by a mechanism distinct from that used by vß5.

FMDV is one of the most infectious animal pathogens known. It causes a severe vesicular disease of cloven-hoofed animals, which spreads by aerosol, sometimes over long distances. In vivo, FMDV shows a strong tropism for epithelial cells. The primary site of infection is thought to be epithelial cells in the upper respiratory tract (3, 10, 11, 14, 51), and during the development of disease, the virus is widely disseminated throughout the body, with secondary sites of replication in many epithelial tissues (3, 12, 13, 14). The ability of FMDV to use multiple, different integrin species to initiate infection could, in part, account for the great success of this virus. However, although integrins are believed to be the receptors used to initiate FMDV infection in an animal, presently we do not know which, if any, of the integrins identified in vitro function in this way. Similarly, very little is known of the tissue distribution and cell type expression of integrins in the natural hosts of FMDV. Studies of other mammalian species have shown that vß3 normally predominates in endothelial rather than epithelial cells (9, 18, 20, 26, 46), whereas, by contrast, vß6 is expressed exclusively in the latter cell type. Recently, vß8 has also been identified on airway epithelial cells (15, 23). If these observations were repeated in the natural hosts of FMDV, they would point to an important role for vß6 and vß8 in the tropism and pathogenesis of FMDV during the initial phase of infection.

 
We thank M. Pitkeathly and S. Shah for the peptides.

This work was supported by DEFRA (project SE2717).

 
* Corresponding author. Mailing address: Pirbright Laboratory, Institute for Animal Health, Ash Rd., Pirbright, Surrey GU24 0NF, United Kingdom. Phone: 44 1483-232441. Fax: 44 1483-237161. E-mail: terry.jackson{at}bbsrc.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Acharya, R., E. Fry, D. Stuart, G. Fox, D. Rowlands, and F. Brown. 1989. The three-dimensional structure of foot-and-mouth disease virus at 2.9 Å resolution. Nature 337:709-716.[CrossRef][Medline]
2
2. Agrez, M., A. Chen, R. I. Cone, R. Pytela, and D. Sheppard. 1994. The vß6 integrin promotes proliferation of colon carcinoma cells through a unique region of the ß6 cytoplasmic domain. J. Cell Biol. 127:545-556.
3
3. Alexandersen, A., M. B. Oleksiewicz, and A. I. Donaldson. 2001. The early pathogenesis of foot-and-mouth disease virus in pigs infected by contact: a quantitative time-course study using TaqMan RT-PCR. J. Gen. Virol. 82:747-755.[Abstract/Free Full Text]
4
4. Baranowski, E., C. M. Ruiz-Jarabo, N. Sevilla, D. Andreu, E. Beck, and E. Domingo. 2000. Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage. J. Virol. 74:1641-1647.[Abstract/Free Full Text]
5
5. Baranowski, E., N. Sevilla, N. Verdaguer, C. M. Ruiz-Jarabo, E. Beck, and E. Domingo. 1998. Multiple virulence determinants of foot-and-mouth disease virus in cell culture. J. Virol. 72:6362-6372.[Abstract/Free Full Text]
6
6. Baxt, B., and Y. Becker. 1990. The effect of peptides containing the arginine-glycine aspartic acid sequence on the adsorption of foot-and-mouth disease virus to tissue culture cells. Virus Genes 4:73-83.[CrossRef][Medline]
7
7. Berinstein, A., M. Roivainen, T. Hovi, P. W. Mason, and B. Baxt. 1995. Antibodies to the vitronectin receptor (integrin vß3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells. J. Virol. 69:2664-2666.[Abstract]
8
8. Blystone, S. D., M. P. Williams, S. E. Slater, and E. J. Brown. 1997. Requirement of integrin ß3 tyrosine 747 for ß3 tyrosine phosphorylation and regulation of vß3 avidity. J. Biol. Chem. 272:28757-28761.[Abstract/Free Full Text]
9
9. Breuss, J. M., N. Gillett, L. Lu, D. Sheppard, and R. Pytela. 1993. Restricted distribution of integrin ß6 mRNA in primate epithelial tissues. J. Histochem. Cytochem. 41:1521-1527.[Abstract]
10
10. Brown, C. C., R. F. Meyer, H. J. Olander, C. House, and C. A. Mebus. 1992. A pathogenesis study of foot-and-mouth disease virus in cattle, using in situ hybridisation. Can. J. Vet. Res. 56:189-193.[Medline]
11
11. Brown, C. C., H. J. Olander, and R. F. Meyer. 1991. A preliminary study of the pathogenesis of foot-and-mouth disease virus, using in situ hybridisation. Vet. Pathol. 28:216-222.[Abstract]
12
12. Brown, C. C., H. J. Olander, and R. F. Meyer. 1995. Pathogenesis of foot-and-mouth disease virus in swine, studied by in-situ hybridisation. J. Comp. Pathol. 113:51-58.[CrossRef][Medline]
13
13. Brown, C. C., M. E. Piccone, P. W. Mason, T. S.-C. McKenna, and M. J. Grubman. 1996. Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. J. Virol. 70:5638-5641.[Abstract/Free Full Text]
14
14. Burrows, R.,. J. A. Mann, A. J. M. Garland, A. Greig, and D. Goodridge. 1981. The pathogenesis of natural and stimulated natural foot-and-mouth disease virus infection in cattle. J. Comp. Pathol. 91:599-609.[CrossRef][Medline]
15
15. Cambier, S., D. Mu, D. O'Connell, K. Boylen, W. Travis, W. Liu, V. C. Broaddus, and S. L. Nishimura. 2000. A role for the integrin vß8 in the negative regulation of epithelial cell growth. Can. Res. 60:7084-7093.[Abstract/Free Full Text]
16
16. Cone, R. I., A. Weinacker, A. Chen, and D. Sheppard. 1994. Effects of ß subunit cytoplasmic domain deletions on the recruitment of the integrin vß6 to focal contacts. Cell Adhesion Commun. 2:101-113.[Medline]
17
17. Curry, S., E. Fry, W. E. Blakemore, R. Abu-Ghazaleh, T. Jackson, A. King, S. Lea, J. Newman, D. Rowlands, and D. Stuart. 1996. Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity. Structure 4:135-145.[Medline]
18
18. Damjanovich, L., S. M. Albelda, S. A. Mette, and C. A. Buck. 1992. Distribution of integrin cell adhesion receptors in normal and malignant lung tissue. Am. J. Respir. Cell Mol. Biol. 6:197-206.
19
19. Dedhar, S., and G. E. Hannigan. 1996. Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr. Opin. Cell Biol. 8:657-669.[CrossRef][Medline]
20
20. Delporte, C., R. S. Redman, and B. J. Baum. 1997. Relationship between the cellular distribution of the vß3/5 integrins and adenoviral infection in salivary glands. Lab. Investig. 77:167-173.[Medline]
21
21. Duque, H., and B. Baxt. 2003. Foot-and-mouth disease virus receptors: comparison of bovine v integrin utilization by type A and O viruses. J. Virol. 77:2500-2511.[Abstract/Free Full Text]
22
22. Escarmis, C., E. C. Carrillo, M. Ferrer, J. F. G. Arriaza, N. Lopez, C. Tami, N. Verdaguer, E. Domingo, and M. T. Franze-Fernandez. 1998. Rapid selection in modified BHK-21 cells of a foot-and-mouth disease virus variant showing alterations in cell tropism. J. Virol. 72:10171-10179.[Abstract/Free Full Text]
23
23. Fjellbirkeland, L., S. Cambier, V. C. Broaddus, A. Hill, P. Brunetta, G. Dolganov, D. Jablons, and S. L. Nishimura. 2003. Integrin vß8-mediated activation of transforming growth factor-ß inhibits human airway epithelial proliferation in intact bronchial tissue. Am. J. Pathol. 163:533-542.[Abstract/Free Full Text]
24
24. Fox, G., N. R. Parry, P. V. Barnett, B. McGinn, D. J. Rowlands, and F. Brown. 1989. Cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). J. Gen. Virol. 70:625-637.[Abstract/Free Full Text]
25
25. Giancotti, F. G., and E. Ruoslahtil. 1999. Integrin signalling. Science 285:1028-1032.[Abstract/Free Full Text]
26
26. Haapasalmi, K., K. Zhang, M. Tonnesen, J. Olerud, D. Sheppard, T. Salo, R. Krammer, R. Clark, V. Uitto, and H. Larjava. 1996. Keratinocytes in human wounds express vß6 integrin. J. Investig. Dermatol. 106:42-48.[CrossRef][Medline]
27
27. Huang, X., J. F. Wu, S. Spong, and D. Sheppard. 1998. The integrin vß6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin. J. Cell Sci. 111:2189-2195.[Abstract]
28
28. Hynes, R. O. 1992. Integrins: versatility, modulation, and signalling in cell adhesion. Cell 69:11-25.[CrossRef][Medline]
29
29. Jackson, T., F. M. Ellard, R. Abu-Ghazaleh, S. M. Brookes, W. E. Blakemore, A. H. Corteyn, D. I. Stuart, J. W. I. Newman, and A. M. Q. King. 1996. Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J. Virol. 70:5282-5287.[Abstract/Free Full Text]
30
30. Jackson, T., A. M. Q. King, D. I. Stuart, and E. Fry. 2003. Structure and receptor binding. Virus Res. 91:33-46.[CrossRef][Medline]
31
31. Jackson. T., A. P. Mould, D. Sheppard, and A. M. Q. King. 2002. The integrin vß1 is a receptor for foot-and-mouth disease virus. J. Virol. 76:935-941.[Abstract/Free Full Text]
32
32. Jackson, T., A. Sharma, R. Abu-Ghazaleh, W. E. Blakemore, F. M. Ellard, D. L. Simmons, J. W. I. Newman, D. I. Stuart, and A. M. Q. King. 1997. Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease virus to the purified integrin vß3 in vitro. J. Virol. 71:8357-8361.[Abstract]
33
33. Jackson, T., D. Sheppard, M. Denyer, W. E. Blakemore, and A. M. Q. King. 2000. The epithelial integrin vß6 is a receptor for foot-and-mouth disease virus. J. Virol. 74:4949-4956.[Abstract/Free Full Text]
34
34. Kirchhofer, D., J. Grzesiak, and M. D. Pierschbacher. 1991. Calcium as a potential physiological regulator of integrin-mediated cell adhesion. J. Biol. Chem. 266:4471-4477.[Abstract/Free Full Text]
35
35. Knowles, N. J., and A. R. Samuel. 2003. Molecular epidemiology of foot-and-mouth disease virus. Virus Res. 91:65-80.[CrossRef][Medline]
36
36. Lee, J. O., L. A. Bankston, M. A. Arnaout, and R. C. Liddington. 1995. Two conformations of the integrin A-domain (I-domain): a pathway for activation? Structure 3:1333-1340.[Medline]
37
37. Li, R., P. Rieu, D. L. Griffith, D. Scott, and M. A. Arnaout. 1998. Two functional states of the CD11b A-domain: correlations with key features of two Mn²⁺-complexed crystal structures. J. Cell Biol. 143:1523-1534.[Abstract/Free Full Text]
38
38. Liebermann, H., R. Dolling, D. Schmidt, and G. Thalmann. 1991. RGD-containing peptides of VP1 of foot-and-mouth disease virus (FMDV) prevent virus infection in vitro. Acta Virol. 35:90-93.[Medline]
39
39. Logan, D., R. Abu-Ghazaleh, W. E. Blakemore, S. Curry, T. Jackson, A. King, S. Lea, R. Lewis, J. W. I. Newman, N. Parry, D. Rowlands, D. Stuart, and E. Fry. 1993. Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 362:566-568.[CrossRef][Medline]
40
40. Ludbrook, S. B., S. T. Barry, C. J. Delves, and C. M. T. Horgan. 2003. The integrin vß3 is a receptor for the latency-associated peptides of transforming growth factors ß1 and ß3. Biochem. J. 369:311-318.[CrossRef][Medline]
41
41. Martinez, M. A., N. Verdaguer, M. Mateu, and E. Domingo. 1997. Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus. Proc. Natl. Acad. Sci. USA 94:6798-6802.[Abstract/Free Full Text]
42
42. Mason, P. W., E. Rieder, and B. Baxt. 1994. RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway. Proc. Natl. Acad. Sci. USA 91:1932-1936.[Abstract/Free Full Text]
43
43. Mason. P. W., B. Baxt, F. Brown, J. Harber, A. Murdin, and E. Wimmer. 1993. Antibody-complexed foot-and-mouth disease virus, but not poliovirus, can infect cells via the Fc receptor. Virology 192:568-577.[CrossRef][Medline]
44
44. Mateu, M. G., M. Luz Valero, D. Andreu, and E. Domingo. 1996. Systematic replacement of amino acid residues within an Arg-Gly-Asp-containing loop of foot-and-mouth disease virus and effects on cell recognition. J. Biol. Chem. 271:12814-12819.[Abstract/Free Full Text]
45
45. McCahon, D., J. R. Crowther, G. J. Belsham, J. D. A. Kitson, M. Duchesne, P. Have, R. H. Meloen, D. O. Morgan, and F. de Simone. 1989. Evidence for at least four antigenic sites on type O foot-and-mouth disease virus involved in neutralization; identification by single and multiple site monoclonal antibody-resistant mutants. J. Gen. Virol. 70:639-645.[Abstract/Free Full Text]
46
46. Mette, S. A., J. Pilewski, C. A. Buck, and S. M. Albelda. 1993. Distribution of integrin cell adhesion receptors in normal bronchial epithelial cells and lung cancer cells in vitro and in vivo. Am. J. Respir. Cell Mol. Biol. 8:562-572.
47
47. Miller, L. C., W. E. Blakemore, D. Sheppard, A. Atakilit, A. M. Q. King, and T. Jackson. 2001. Role of the cytoplasmic domain of the ß-subunit of integrin vß6 in infection by foot-and-mouth disease virus. J. Virol. 75:4158-4164.[Abstract/Free Full Text]
48
48. Mould, A. P., S. K. Akiyama, and M. J. Humphries. 1995. Regulation of integrin 5ß1-fibronectin interactions by divalent cations. J. Biol. Chem. 270:26270-26277.[Abstract/Free Full Text]
49
49. Mu, D., S. Cambier, L. Fjellbirkeland, J. L. Baron, J. S. Munger, H. Kawakatsu, D. Sheppard, V. C. Broaddus, and S. L. Nishimura. 2002. The integrin vß8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-ß1. J. Cell Biol. 157:493-507.[Abstract/Free Full Text]
50
50. Munger, J. S., X. Huang, H. Kawakatsu, M. D. J. Griffiths, S. L. Dalton, J. Wu, J. F. Pittet, N. Kaminski, C. Garat, M. A. Matthay, D. B. Rifkin, and D. Sheppard. 1999. The integrin vß6 binds and activates latent TGFß1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96:319-328.[CrossRef][Medline]
51
51. Murphy, P. M. L., M. A. Forsyth, G. J. Belsham, and J. S. Salt. 1999. Localization of foot-and-mouth disease virus RNA by in situ hybridisation within bovine tissues. Virus Res. 62:67-76.[CrossRef][Medline]
52
52. Neff, S., D. Sa-Carvalho, E. Rieder, P. W. Mason, S. D. Blystone, E. J. Brown, and B. Baxt. 1998. Foot-and-mouth disease virus virulent for cattle utilizes the integrin vß3 as its receptor. J. Virol. 72:3587-3594.[Abstract/Free Full Text]
53
53. Neff, S., and B. Baxt. 2001. The ability of integrin vß3 to function as a receptor for foot-and-mouth disease virus is not dependent on the presence of complete subunit cytoplasmic domains. J. Virol. 75:527-532.[Abstract/Free Full Text]
54
54. Nishimura, S. L., D. Sheppard, and R. Pytela. 1994. Integrin vß8. Interaction with vitronectin and functional divergence of the ß8 cytoplasmic domain. J. Biol. Chem. 269:28708-28715.[Abstract/Free Full Text]
55
55. O'Toole, T. E., J. Ylanne, and B. M. Culley. 1995. Regulation of integrin affinity states through an NPXY motif in the ß subunit cytoplasmic domain. J. Biol Chem. 270:8553-8558.[Abstract/Free Full Text]
56
56. Pfaff, E., H.-J. Thiel, E. Beck, K. Strohmaier, and H. Schaller. 1988. Analysis of neutralizing epitopes on foot-and-mouth disease virus. J. Virol. 62:2033-2040.[Abstract/Free Full Text]
57
57. Prieto, A. L., G. M. Edelman, and K. L. Crossin. 1993. Multiple integrins mediate cell attachment to cytotactin/tenascin. Proc. Natl. Acad. Sci. USA 90:10154-10158.[Abstract/Free Full Text]
58
58. Sa-Carvalho, D., E. Rieder, B. Baxt, R. Rodarte, A. Tanuri, and P. W. Mason. 1997. Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. J. Virol. 71:5115-5123.[Abstract]
59
59. Surovoi, A. Y., V. T. Ivanov, A. V. Chepurkin, V. N. Ivanyuschenkov, and N. N. Dryagalin. 1988. Is the Arg-Gly-Asp sequence the site for foot-and-mouth disease virus binding with cell receptor? Sov. J. Bioorg. Chem. 14:965-968.
60
60. Van Nhieu, G. T., E. S. Krukonis, A. A. Reszka, A. F. Horwitz, and R. R. Isberg. 1996. Mutations in the cytoplasmic domain of the integrin ß1 chain indicate a role for endocytosis factors in bacterial internalisation. J. Biol. Chem. 271:7665-7672.[Abstract/Free Full Text]
61
61. Wang, K., T. Guan, D. A. Cheresh, and G. R. Nemerow. 2000. Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin ß5. J. Virol. 74:2731-2739.[Abstract/Free Full Text]
62
62. Weinacker, A., A. Chen, M. Agrez, R. I. Cone, S. Nishimura, E. Wayner, R. Pytela, and D. Sheppard. 1994. Role of the integrin vß6 in cell attachment to fibronectin. J. Biol. Chem. 269:6940-6948.[Abstract/Free Full Text]
63
63. Ylänne, J., J. Huuskonen, T. E. O'Toole, M. H. Ginsberg, I. Virtanen, and C. G. Gahmberg. 1995. Mutation of the cytoplasmic domain of the integrin ß3 subunit. Differential effects on cell spreading, recruitment to adhesion plaques, endocytosis and phagocytosis. J. Biol. Chem. 270:9550-9557.[Abstract/Free Full Text]
64
64. Yokosaki, Y., H. Monis, J. Chen, and D. Sheppard. 1996. Differential effects of the integrins 9ß1, vß3 and vß6 on cell proliferative responses to tenascin. J. Biol. Chem. 271:24144-24150.[Abstract/Free Full Text]
65
65. Zhao, Q., J. M. Pacheco, and P. W. Mason. 2003. Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals. J. Virol. 77:3269-3280.[Abstract/Free Full Text]

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Journal of Virology, May 2004, p. 4533-4540, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4533-4540.2004
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• Burman, A., Clark, S., Abrescia, N. G. A., Fry, E. E., Stuart, D. I., Jackson, T. (2006). Specificity of the VP1 GH Loop of Foot-and-Mouth Disease Virus for {alpha}v Integrins. J. Virol. 80: 9798-9810 [Abstract] [Full Text]  
• Pellinen, T., Ivaska, J. (2006). Integrin traffic.. J. Cell Sci. 119: 3723-3731 [Abstract] [Full Text]  
• Brown, J. K., McAleese, S. M., Thornton, E. M., Pate, J. A., Schock, A., Macrae, A. I., Scott, P. R., Miller, H. R.P., Collie, D. D.S. (2006). Integrin-{alpha}v{beta}6, a Putative Receptor for Foot-and-Mouth Disease Virus, Is Constitutively Expressed in Ruminant Airways. J. Histochem. Cytochem. 54: 807-816 [Abstract] [Full Text]  
• Rieder, E., Henry, T., Duque, H., Baxt, B. (2005). Analysis of a Foot-and-Mouth Disease Virus Type A24 Isolate Containing an SGD Receptor Recognition Site In Vitro and Its Pathogenesis in Cattle. J. Virol. 79: 12989-12998 [Abstract] [Full Text]  
• Monaghan, P., Gold, S., Simpson, J., Zhang, Z., Weinreb, P. H., Violette, S. M., Alexandersen, S., Jackson, T. (2005). The {alpha}v{beta}6 integrin receptor for Foot-and-mouth disease virus is expressed constitutively on the epithelial cells targeted in cattle. J. Gen. Virol. 86: 2769-2780 [Abstract] [Full Text]  
• Fry, E. E., Newman, J. W. I., Curry, S., Najjam, S., Jackson, T., Blakemore, W., Lea, S. M., Miller, L., Burman, A., King, A. M. Q., Stuart, D. I. (2005). Structure of Foot-and-mouth disease virus serotype A1061 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J. Gen. Virol. 86: 1909-1920 [Abstract] [Full Text]  
• O'Donnell, V., LaRocco, M., Duque, H., Baxt, B. (2005). Analysis of Foot-and-Mouth Disease Virus Internalization Events in Cultured Cells. J. Virol. 79: 8506-8518 [Abstract] [Full Text]  
• Berryman, S., Clark, S., Monaghan, P., Jackson, T. (2005). Early Events in Integrin {alpha}v{beta}6-Mediated Cell Entry of Foot-and-Mouth Disease Virus. J. Virol. 79: 8519-8534 [Abstract] [Full Text]  
• Duque, H., LaRocco, M., Golde, W. T., Baxt, B. (2004). Interactions of Foot-and-Mouth Disease Virus with Soluble Bovine {alpha}V{beta}3 and {alpha}V{beta}6 Integrins. J. Virol. 78: 9773-9781 [Abstract] [Full Text]  

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