Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection.Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD؉1 and RGD؉4 sites) as key for inhibition of virus binding and infection mediated by ␣v␤6 or ␣v␤8 but not for inhibition of virus binding to ␣v␤3. We also show that FMDV peptides containing either methionine or arginine at the RGD؉1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for ␣v␤6. In contrast, although RGDM-containing peptides were effective for ␣v␤8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses ␣v␤8 less efficiently than ␣v␤6 as a receptor for infection. Finally, evidence is presented that shows ␣v␤3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to ␣v␤6 with a higher affinity than to ␣v␤3 and ␣v␤8.Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease, a severe vesicular disease of cloven-hoofed animals, including cattle, sheep, goats, and pigs. The virus exists as seven serotypes, which are members of the genus Aphthovirus of the family Picornaviridae. The type O and A viruses have the broadest geographical distribution and occur in many parts of the world, including Africa, southern Asia, the Far East, and South America (25).The mature virus particle consists of 60 copies (each) of four virus-encoded structural proteins, VP1 to VP4. These proteins form an icosahedral capsid that encloses a single-stranded positive-sense RNA genome. A major structural feature of the capsid is a surface-exposed, conformationally flexible loop: the GH loop of VP1 (1, 30). Synthetic peptides corresponding to this loop inhibit FMDV binding and infection of cells in culture (4,17,29,50), are highly immunogenic, and induce high levels of neutralizing antibody (8,41,42). In all of the FMD viruses studied, the VP1 GH loop is structurally disordered (1, 13, 27). For the type O viruses this has been attributed, in part, to the presence of a disulfide bond tethering one end of the loop to VP2. Crystallographic analysis of chemically reduced virus (44), in which this disulfide was broken, resulted in the loop adopting predominantly one conformation. In this structure the loop lies in a small depression on the surface of the capsid and is formed by a short region of ␤-strand, followed by an arginine-glycine-aspartic acid (RGD) tripeptide in ...

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three ␣v integrins, ␣v␤1, ␣v␤3, and ␣v␤6, 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 ␤8 cDNA and expression of ␣v␤8 at the cell surface. The involvement of ␣v␤8 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 ␣v␤8 heterodimer or the ␣v chain. Similar results were obtained with a chimeric ␣v␤8 including the ␤6 cytodomain (␣v␤8/6), showing that the ␤6 cytodomain can substitute efficiently for the corresponding region of ␤8. In contrast, virus binding to ␣v␤6 including the ␤8 cytodomain (␣v␤6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the ␣v␤6 chimera was recognized poorly by antibodies specific for the ectodomain of ␣v␤6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the ␤6 cytodomain is important for maintaining ␣v␤6 in a conformation required for productive infection by FMDV.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 lowand high-affinity states, enab...

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin ␣v␤6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominantnegative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of ␣v␤6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with ␣v␤6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37°C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin ␣v␤6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin ␣v␤6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD ؉1 and RGD ؉4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to ␣v␤6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin ␣v␤6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.Foot-and-mouth disease virus (FMDV) is the type species of the genus Aphthovirus within the family Picornaviridae and the etiological agent of foot-and-mouth disease, a severe vesicular condition affecting a large number of artiodactyls, including domesticated ruminants and pigs (1, 34). Presently, the virus is endemic in many parts of the world, including South America, Africa, and Asia (19). Foot-and-mouth disease is highly contagious and difficult to control as FMDV has a wide host range (see above) and a rapid replication cycle, small amounts of virus can initiate infection, and infected animals excrete high levels of virus. In addition, multiple modes of transmission have been recognized, including airborne spread, sometimes over long distances, including overseas (1,10,12,31).Field isolates of FMDV use integrins to initiate infection (14, 15, 29). The integrin family of cell adhesion receptors are a conserved series of ␣␤ heterodimers, which bind in a divalent cation-dependent manner to ligands through recognition of short motifs that usually include one of the acidic residues glutamate (E) or aspartate (D) (13). Examples of such motifs include arginine-glycine-aspartate (RGD) or leucine-aspartate-valine (LDV), and short peptides containing these motifs can interact similarly with integrins (13). Recognition of RGDcontaining proteins can proceed in a stepwise manner where the initial RGD binding is enhanced by a second stabilizing interaction involving so-called synergy sites on the ligand (2, 21, 23). The concept of a synergy site was first described for binding of ␣5␤1 to fibronectin (Fn). Thus, high-affinity binding of Fn to ␣5␤1 requires the RGD motif located on the 10th type III domain of Fn and a second synergy site in the 9th type III domain (23). Similarly, the large extracellular matrix protein ...

Field isolates of foot-and-mouth disease virus (FMDVF oot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most widespread, epizootic transboundary animal diseases, affecting many species of wildlife and livestock, such as cattle, sheep, goats, and pigs. The significant economic losses that result from FMD are due to the high morbidity of infected animals and stringent trade restrictions imposed on affected countries (1). Vaccination plays a major role in controlling FMD, either to lessen the effects of an outbreak in FMDfree countries or for control and eradication in regions where it is endemic. The etiological agent of FMD, foot-and-mouth disease virus (FMDV), exists as seven distinct serotypes (O, A, C, Asia-1, and the Southern African Territories [SAT] serotypes SAT-1, SAT-2, and SAT-3). Within each serotype, a large number of antigenic variants exist (2). Intraserotype diversity is driven by a high mutation rate during replication that is caused by an error-prone viral RNA-dependent RNA polymerase (3) and thus complicates efforts to control disease by vaccination due to incomplete protection between some antigenic variants (4). Hence, the most effective vaccines closely match the outbreak virus, which can necessitate the development of new vaccine strains. The current vaccines are inactivated virus preparations grown in large-scale cell culture. Therefore, the production of a new vaccine is critically dependent upon adaptation of viruses from the field for growth in cell culture, which can prove problematical for some viruses.Foot-and-mouth disease virus is the type species of the Aphthovirus genus of the Picornaviridae, a family of nonenveloped, single-stranded positive-sense RNA viruses. The viral capsid is formed by 60 copies each of four structural proteins (VP1 to VP4) arranged in icosahedral symmetry. The outer capsid surfaces are formed by VP1, which surrounds the five-fold symmetry axis, and VP2 and VP3, which alternate around the three-fold axis (5). VP4 is myristoylated and located inside the capsid and is thought to play an essential role in the final stage of assembly and in endosomal membrane penetration by the viral RNA (6, 7). In vivo, FMDV has a strong tropism for epithelial cells, which is in part due to the epithelial cell-restricted expression of integrin ␣v␤6, which is the principal receptor used by field viruses to initiate infection (8-12). Integrin binding is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) motif located at the apex of a structurally disordered loop (the GH loop of VP1). The integrin specificity of FMDV has been the subject of several studies, and three other RGD-dependent integrins (␣v␤1, ␣v␤3, and ␣v␤8) have also been reported to be receptors for field strains of the virus (13-15); however, the role of these integrins in pathogenesis is unclear, and we have found that ␣v␤3 is a poor receptor for FMDV in vitro (16). Furthermore, despite recognizing their ligands via the RGD motif, two other RGD-dependent integrins ...

We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I 248 3T in 2C, Q 44 3R in 3A, and L 147 3P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L 147 3P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L 147 3P, and this infection was inhibited by antibodies to ␣v␤6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin ␣v␤6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T 248 3N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species.The high potential for adaptation and rapid evolution that derives from the quasispecies dynamics of RNA virus populations (30, 40) can be reflected in the alteration of cell tropism, host range, and virulence (8,9,30,43,51). Mutant viruses with a modified host range can contribute to the emergence of new animal and human diseases (62). On the other hand, adaptation to a new host has been exploited since the beginning of vaccinology to derive attenuated strains with decreased pathogenicity for the original, natural host (18).Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is the etiological agent of the most important animal disease affecting domestic cloven-hoofed animals and a large variety of wild artiodactyls (for reviews, see references 2, 6, 21, 63, 68, and 74). The virus consists of a nonenveloped particle of icosahedral symmetry containing a positive-sense single-stranded RNA genome of about 8.5 kb. A single open reading frame encodes all of the capsid, as well as a total of nine additional mature, nonstructural (NS) proteins, including two proteases (L and 3C) and an RNA-dependent RNA polymerase (3D) (14,69,73). As shown for a number of different picornaviruses, the mature NS proteins, as well as some of their protein precursors, are involved in multiple functions needed for virus multiplication and the host cell membrane rearrangements associated with viral RNA replication (3,25,35,46,55,60,65,82).FMDV can initiate the infection of cultured cells via different ␣v integrins (␣v␤1,␣v␤...

The iron uptake systems of pathogenic bacteria provide potential targets for immunological intervention. We have partially purified the high molecular mass, iron-regulated outer membrane proteins (IROMPs) from Pseudornonas aeruginosu and used them to prepare a panel of monoclonal antibodies (mAbs). Five mAbs reacted with an 85 kDa IROMP separated by SDS-PAGE, but gave only low-level binding to whole cells by immunogold electron microscopy. However, iodination of whole cells indicated that the 85 kDa IROMP is surface-exposed. The mAbs were only cross-reactive with clinical isolates representing eight of the 17 International Antigenic Typing Scheme serotypes of P. aeruginosa, suggesting significant heterogeneity with respect to this IROMP.

The potential of a new HAT-sensitive human lymphoblastoid cell line TK6 TG(r).P1. as a fusion partner was assessed, by comparison with the established human parental cell line UC729.6. Both of these cell lines were fused with the peripheral blood mononuclear cells of a patient with B-chronic lymphocytic leukaemia. The hybridomas generated in these fusion experiments were analysed by the fluorescence activated cell sorter and karyotyping. An anti-idiotype ELISA assay detected the presence of the patient's characteristic idiotype bearing immunoglobulin in the supernatant of a number of the hybridoma cell lines generated in both fusions.