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Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus
Virus Research 62 (1999) 169 – 175 www.elsevier.com/locate/virusres
Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus E. Domingo a,*, N. Verdaguer b, W.F. Ochoa b, C.M. Ruiz-Jarabo a, N. Sevilla a,1, E. Baranowski a, M.G. Mateu a, I. Fita b a
Centro de Biologı´a Molecular ‘Se6ero Ochoa’, Uni6ersidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain b Centre de In6estigacio´ i Desen6olupament (CSIC), Jordi Girona 6, 08028 Barcelona, Spain
Abstract The function of a loop exposed on the aphthovirus capsid (the G – H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Foot-and-mouth disease virus; Neutralizing antibodies; Picornaviral disease
1. Introduction Neutralizing antibodies are important determinants of protection against picornaviral disease (McCullough et al., 1992; Misbah et al., 1992; Mateu, 1995). A combination of genetic, immunochemical and structural approaches has been used for the identification and characterization of anti* Corresponding author. Tel.: +34-91-3978485; fax: + 3491-3974799. E-mail address: [email protected] (E. Domingo) 1 Present address: The Scripps Research Institute, Department of Neuropharmacology (IMM-6), 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
genic sites on the picornaviral particle (Minor, 1990; Mateu, 1995; Usherwood and Nash, 1995). Studies in our laboratories with the animal pathogen foot-and-mouth disease virus (FMDV), an aphthovirus of the Picornaviridae family, have been aimed at understanding the structural basis of antigen–antibody interactions, and the mechanisms of virus escape from neutralization by antibodies (Mateu, 1995). The experiments have involved FMDV clone C-S8c1 (a plaque-purified virus derived from the natural isolate C-StaPau Sp/70; Sobrino et al., 1983), a number of natural isolates, laboratory-adapted viruses related to CS8c1, and a collection of monoclonal antibodies (MAbs) raised against FMDV of serotype C (Ma-
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teu et al. 1987, 1990). Based on the mapping on the three-dimensional structure of C-S8c1 of amino acid replacements in MAb-resistant (MAR) mutants of C-S8c1, and assays of reactivity of MAbs with synthetic peptides, two distinct antigenic sites involved in neutralization of CS8c1 were identified (Lea et al., 1994). The two sites, termed A and D, are immunodominant in natural hosts for FMDV, and include multiple epitopes (Mateu et al., 1989, 1990; Lea et al., 1994; Mateu et al., 1994; Mateu, 1995; Holguı´n et al., 1997). Antigenic site D is composed of discontinuous epitopes involving the three exposed capsid proteins VP1, VP2 and VP3 (Lea et al., 1994). Discontinuous epitopes are difficult to mimic with synthetic peptides. However, an unprocessed capsid precursor (P1) expressed in mammalian cells acquired at least some of the site D epitopes, thus providing a means to study the interaction of such discontinuous epitopes with antibodies (Mateu et al., 1998). In contrast to site D, antigenic site A is composed of continuous, partially overlapping epitopes located within the G-H loop of capsid protein VP1. This loop includes a highly conserved Arg-Gly-Asp (RGD) triplet (positions 141 – 143) involved in recognition of an integrin receptor (Fox et al., 1989; Berinstein et al., 1995; Mason et al., 1994; Jackson et al., 1997). Synthetic peptides spanning residues 133 – 156 of VP1 faithfully mimic the entire virus particle with regard to interaction with antibodies and also recognition of an integrin receptor (Mateu, 1995; Mateu et al., 1996). The G-H loop of capsid protein VP1 is disordered in crystals of FMDV particles (Acharya et al., 1989; Lea et al., 1994, 1995; Curry et al., 1996). A structure could be defined on crystals of chemically reduced FMDV O1BFS particles (Logan et al., 1993). The loop was highly structured, and bent towards the three-fold axis of the particle in an orientation that has been called the ‘down’ position. Interestingly, a similar loop structure was found in a complex between an antigenic peptide representing residues 136 – 150 of the G-H loop of VP1 of C-S8c1 and the Fab fragment of each of two neutralizing MAbs, SD6 and 4C4, raised against the virus (Verdaguer et al., 1995, 1998). In these two complexes the loop
acquired a quasi-circular shape, very similar to the structure found by Logan et al., (1993) with FMDV of serotype O. Comparison of the structures of the Fabs alone with those of the corresponding complexes indicated that the antibodies underwent considerable induced fit upon binding to the peptide antigen (Verdaguer et al., 1996). The structures of the two antibodies became more similar to accommodate to the peptide, and both used a similar array of interactions (Verdaguer et al., 1998). In the peptide antigen the RGD triplet, in particular D-143, played a pivotal role in the interaction with the two antibodies (Verdaguer et al., 1998). Moreover, measurements of reactivities of substituted peptides with additional site A-specific MAbs suggest that other MAbs may also interact through the RGD and neighboring residues with the antigenic peptide (Verdaguer et al., 1998). The similarity of the structures of a synthetic peptide antigen in a complex with two Fab fragments and the loop in reduced FMDV O1 (compare Logan et al., 1993 with Verdaguer et al. 1995, 1998), together with evidence from proton 2 H nuclear magnetic resonance (NMR) spectroscopy with cyclic antigenic peptides in solution (Haack et al., 1997), reinforces the notion that the loop may exist as a stable, quasi-circular structure in FMDV particles. Its delocalization in native FMDV would probably result from some hinge movement about the anchor points of the loop on the virion surface. This is also suggested by cryoelectron microscopy studies of complexes between the Fab of MAbs SD6 and 4C4 with FMDV C-S8c1 (Hewat et al., 1997; Hewat et al., submitted for publication). However, the evidence of the loop structure in FMDV of serotype C was derived from complexes with Fab employing a single peptide antigen, the one that corresponds to the amino acid sequence of C-S8c1. More recent results with substituted antigenic peptides (Ochoa et al., in preparation) reinforce the occurrence of a quasi circular G-H loop structure for VP1, with the structural features previously described by Logan et al. (1993) and Verdaguer et al. (1995, 1998).
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2. Unusual variants and the compromise between receptor recognition and antibody escape RNA viruses adapt by producing many mutants (in the order of one mutation in each molecule of progeny RNA copied from a template) and expanding those mutants which are most fit in the particular environment in which replication takes place (recent review in Domingo and Holland, 1997). Antigenic site A of FMDV constitutes an excellent model system to explore with molecular detail how a virus uses the same site for two seemingly contradictory purposes. One is receptor recognition, a function expected to dictate constancy of the viral sequences involved. The other is escape from antibodies which dictates variation. Part of the problem posed by these conflicting demands was solved by postulating that receptor recognition sites on the virus would be hidden from immune attack, and that they would be physically separated from the sites recognized by antibodies. However, the work with FMDV (Verdaguer et al., 1995, 1998), poliovirus (Harber et al., 1995) and rhinovirus (Smith et al., 1996) has documented that residues belonging to antigenic sites may be also involved in receptor recognition. In the case of FMDV the critical Arg-Gly-Asp triplet is involved both in integrin recognition and antibody binding. This multifunctionality should not be surprising since RNA genomes have evolved a number of molecular designs to accumulate multiple information in a limited genomic region: overlapping open-reading frames, ambisense RNA, multifunctional proteins, among other devices. Yet in all cases mutant generation and potential for rapid variation are successfully exploited for adaptation of RNA viruses (Domingo et al., 1993, 1996). In the specific case provided by site A of FMDV the solution to this riddle is being provided by new observations on receptor usage and, probably related to the latter, evidence of dispensability of residues once thought to be strictly required for cell recognition. A key observation for FMDV, and recently extended to other viruses, was that cell culture adaptation paralleled the ability of the virus to bind hep-
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arin, and to use binding to heparan sulfate as a step necessary for infection (Jackson et al., 1996; Sa-Carvalho et al., 1997). This cell tropism alteration involves acquisition by mutation of positively charged amino acids at the capsid surface, although the location of such critical residues appears to vary for different FMDV strains (Baranowski et al., 1998). It is not yet entirely clear whether heparan sulfate can act as a receptor on its own, or as a coreceptor, or simply as a first landing site as a step towards internalization by a bona fide receptor, an integrin or other. In the course of studies aimed at identifying variations in the FMDV C-S8c1 capsid that could be associated with gains in relative fitness of the virus in BHK-21 cells, we isolated a number of mutants resistant to MAb SD6. None out of 82 escape mutants of FMDV C-S8c1 included variations at the RGD triplet while, surprisingly, five out of 31 escape mutants of FMDV C-S8c1 passaged one hundred times in BHK-21 cells had substitutions at the RGD (Martı´nez et al., 1997). One of the mutants included an RGG (instead of RGD) triplet, and a number of additional capsid replacements, with acquisition of positively charged amino acids on its surface. This virus, as well as other mutants of the same passage series, acquired weak binding for heparin— a binding which is not observed with the parental C-S8c1 FMDV—and the ability to infect Chinese hamster ovary (CHO) cells. However, mutant CHO cells deficient in heparan sulfate could also be infected (Baranowski et al., 1998). This rather puzzling observation is now being investigated further. Recent experiments have documented a considerable genetic stability of the RGG mutant in cell culture. Also, this mutant can be effectively neutralized by polyclonal antibodies directed to site A of C-S8c1 (Ruiz-Jarabo et al., 1999). Thus the RGD triplet can be dispensable for efficient infection by FMDV of cells in culture. The studies of Mateu et al. (1996) and of Verdaguer et al. (1998) allow a comparison of the effects of amino acid replacements on a synthetic peptide representing antigenic site A on inhibition of FMDV infectivity and binding to MAbs.
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Although some residues have a different involvement in cell recognition and antibody binding, Gly-142 and Asp-143 of the RGD triplet, and also the neighboring residues Leu-144 and Leu147 play critical roles in the two types of interactions. Therefore, it was unexpected that a viable virus with substitution Asp-143 Gly would be readily obtained (Martı´nez et al., 1997). It is not known whether this mutant is still able to recognize an integrin receptor or it uses alternative receptors for entry into the cell. Evolution of FMDV has shaped site A in a way that it has sufficient tolerance to replacements so as to escape from neutralizing antibodies and still be able to perform the essential receptor recognition and cell entry functions (Domingo et al., 1996). The repeated finding of the same loop structure in a variety of complexes involving different antibodies and peptide antigens (Verdaguer et al., 1995, 1998, 1999; Ferna´ndez et al., manuscript in preparation) suggests that a considerable number of replacements may cause only subtle structural alterations while preserving the overall quasi-cyclical shape of the loop. Furthermore, the recent observations summarized in the present report suggest that loop variations may not only be permissive for integrin recognition but may actually be irrelevant in some cases, in view of the potential use of alternative receptors. This important question is being actively investigated by several groups.
‘down’ position found in reduced FMDV O1 BFS (Logan et al., 1993; Hewat et al. 1997; Verdaguer et al., 1999). These comparisons suggest that the loop may have a hinge movement (Parry et al., 1990) on the virion surface, perhaps contributing to its immunodominance with alternative modes of interaction with antibodies. Both the cryo-electron microscopy and X-ray crystallographic reconstructions, together with biochemical studies of neutralization by the MAbs and their Fab moieties, suggest that the antibodies neutralize by monovalent binding to the loop without aggregation of virions (Verda-
3. Current view of FMDV antigenic site A and neutralization mechanism The structures of peptide – antibody complexes (Verdaguer et al., 1995, 1998) were combined with cryo-electron microscopy studies of the same Fab molecules bound to C-S8c1 particles (Hewat et al., 1997; Verdaguer et al., 1999). This has allowed the positioning of the loop on the virion surface (Fig. 1). Interestingly the present evidence suggests that the position of the loop relative to reference residues on the capsid surface is different when it is bound to MAb SD6 than when it is bound to MAb 4C4 (Verdaguer et al., 1999). Both modeled positions are not coincident with the
Fig. 1. Ribbon diagram of a biological protomer of FMDV C-S8c1. The capsid proteins VP1, VP2 and VP3 are represented in light grey. The position of the G-H loop of VP1 was derived from cryo-electron microscopy reconstructed models of complexes between FMDV C-S8c1 and the Fab fragment of either MAb SD6 or 4C4 (Hewat et al., 1997; Verdaguer et al., 1999). The position of the loop in the SD6 and 4C4 complexes is shown in dark and medium grey, respectively. For comparison, the disposition of the loop determined in the crystallographic structure of the reduced FMDV O1BFS (Logan et al., 1993) is depicted in white. The side-chains of arginine and aspartic acid of the essential Arg-Gly-Asp triplet are shown.
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guer et al., 1997, 1998). Thus the current model is that the family of site A-specific MAbs that we have used to define the epitopes of site A (reviewed in Mateu, 1995) probably neutralize by binding to the loop and by interfering with receptor recognition. Many interesting questions remain. In some multiply passaged FMDV populations, the requirement of an RGD has been relaxed to the point that multiple variants with unusual substitutions at RGD and neighboring residues have been isolated among biological clones of the quasispecies (Ruiz-Jarabo et al., 1999). It appears as if this antigenic site could undergo a rather unrestricted diversification. Implications for FMDV pathogenesis and disease control are obvious. Would the loop maintain a quasi-circular shape (Logan et al., 1993; Verdaguer et al., 1999) when it is modified by multiple amino acid substitutions? What is the antigenic repertoire that should be incorporated in a synthetic vaccine formulation? These, and some related questions, are now under study, not only for academic reasons, but also because a broad repertoire of antibodies able to bind the loop in different manners may help in avoiding selection of FMDV-escape mutants. Thus, cocktails of different versions of antigenic site A peptides, designed according to the results of neutralization of infectivity in cell culture, and according also with the repertoire of escape mutants seen in vivo (Taboga et al., 1997), may help in the design of synthetic vaccines.
Acknowledgements We are indebted to D. Stuart, A. King, E. Hewat and their colleagues for important contributions to structural studies with FMDV, and to E. Brocchi for the generous supply of MAb 4C4. Work in Madrid was supported by grants from DGES PM97-0060-C02-01 and Fundacio´n Ramo´n Areces. Work at CID in Barcelona was supported by grant PB95-0218 from DGICYT. Work at Universitat de Barcelona was supported by grant DGICYT PB94-00845 and by Centre de Refere`ncia de Biotecnologia de la Generalitat de Catalunya. E.B. is a postdoctoral fellow from M.E.C.
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Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus E. Domingo a,*, N. Verdaguer b, W.F. Ochoa b, C.M. Ruiz-Jarabo a, N. Sevilla a,1, E. Baranowski a, M.G. Mateu a, I. Fita b a
Centro de Biologı´a Molecular ‘Se6ero Ochoa’, Uni6ersidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain b Centre de In6estigacio´ i Desen6olupament (CSIC), Jordi Girona 6, 08028 Barcelona, Spain
Abstract The function of a loop exposed on the aphthovirus capsid (the G – H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Foot-and-mouth disease virus; Neutralizing antibodies; Picornaviral disease
1. Introduction Neutralizing antibodies are important determinants of protection against picornaviral disease (McCullough et al., 1992; Misbah et al., 1992; Mateu, 1995). A combination of genetic, immunochemical and structural approaches has been used for the identification and characterization of anti* Corresponding author. Tel.: +34-91-3978485; fax: + 3491-3974799. E-mail address: [email protected] (E. Domingo) 1 Present address: The Scripps Research Institute, Department of Neuropharmacology (IMM-6), 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
genic sites on the picornaviral particle (Minor, 1990; Mateu, 1995; Usherwood and Nash, 1995). Studies in our laboratories with the animal pathogen foot-and-mouth disease virus (FMDV), an aphthovirus of the Picornaviridae family, have been aimed at understanding the structural basis of antigen–antibody interactions, and the mechanisms of virus escape from neutralization by antibodies (Mateu, 1995). The experiments have involved FMDV clone C-S8c1 (a plaque-purified virus derived from the natural isolate C-StaPau Sp/70; Sobrino et al., 1983), a number of natural isolates, laboratory-adapted viruses related to CS8c1, and a collection of monoclonal antibodies (MAbs) raised against FMDV of serotype C (Ma-
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teu et al. 1987, 1990). Based on the mapping on the three-dimensional structure of C-S8c1 of amino acid replacements in MAb-resistant (MAR) mutants of C-S8c1, and assays of reactivity of MAbs with synthetic peptides, two distinct antigenic sites involved in neutralization of CS8c1 were identified (Lea et al., 1994). The two sites, termed A and D, are immunodominant in natural hosts for FMDV, and include multiple epitopes (Mateu et al., 1989, 1990; Lea et al., 1994; Mateu et al., 1994; Mateu, 1995; Holguı´n et al., 1997). Antigenic site D is composed of discontinuous epitopes involving the three exposed capsid proteins VP1, VP2 and VP3 (Lea et al., 1994). Discontinuous epitopes are difficult to mimic with synthetic peptides. However, an unprocessed capsid precursor (P1) expressed in mammalian cells acquired at least some of the site D epitopes, thus providing a means to study the interaction of such discontinuous epitopes with antibodies (Mateu et al., 1998). In contrast to site D, antigenic site A is composed of continuous, partially overlapping epitopes located within the G-H loop of capsid protein VP1. This loop includes a highly conserved Arg-Gly-Asp (RGD) triplet (positions 141 – 143) involved in recognition of an integrin receptor (Fox et al., 1989; Berinstein et al., 1995; Mason et al., 1994; Jackson et al., 1997). Synthetic peptides spanning residues 133 – 156 of VP1 faithfully mimic the entire virus particle with regard to interaction with antibodies and also recognition of an integrin receptor (Mateu, 1995; Mateu et al., 1996). The G-H loop of capsid protein VP1 is disordered in crystals of FMDV particles (Acharya et al., 1989; Lea et al., 1994, 1995; Curry et al., 1996). A structure could be defined on crystals of chemically reduced FMDV O1BFS particles (Logan et al., 1993). The loop was highly structured, and bent towards the three-fold axis of the particle in an orientation that has been called the ‘down’ position. Interestingly, a similar loop structure was found in a complex between an antigenic peptide representing residues 136 – 150 of the G-H loop of VP1 of C-S8c1 and the Fab fragment of each of two neutralizing MAbs, SD6 and 4C4, raised against the virus (Verdaguer et al., 1995, 1998). In these two complexes the loop
acquired a quasi-circular shape, very similar to the structure found by Logan et al., (1993) with FMDV of serotype O. Comparison of the structures of the Fabs alone with those of the corresponding complexes indicated that the antibodies underwent considerable induced fit upon binding to the peptide antigen (Verdaguer et al., 1996). The structures of the two antibodies became more similar to accommodate to the peptide, and both used a similar array of interactions (Verdaguer et al., 1998). In the peptide antigen the RGD triplet, in particular D-143, played a pivotal role in the interaction with the two antibodies (Verdaguer et al., 1998). Moreover, measurements of reactivities of substituted peptides with additional site A-specific MAbs suggest that other MAbs may also interact through the RGD and neighboring residues with the antigenic peptide (Verdaguer et al., 1998). The similarity of the structures of a synthetic peptide antigen in a complex with two Fab fragments and the loop in reduced FMDV O1 (compare Logan et al., 1993 with Verdaguer et al. 1995, 1998), together with evidence from proton 2 H nuclear magnetic resonance (NMR) spectroscopy with cyclic antigenic peptides in solution (Haack et al., 1997), reinforces the notion that the loop may exist as a stable, quasi-circular structure in FMDV particles. Its delocalization in native FMDV would probably result from some hinge movement about the anchor points of the loop on the virion surface. This is also suggested by cryoelectron microscopy studies of complexes between the Fab of MAbs SD6 and 4C4 with FMDV C-S8c1 (Hewat et al., 1997; Hewat et al., submitted for publication). However, the evidence of the loop structure in FMDV of serotype C was derived from complexes with Fab employing a single peptide antigen, the one that corresponds to the amino acid sequence of C-S8c1. More recent results with substituted antigenic peptides (Ochoa et al., in preparation) reinforce the occurrence of a quasi circular G-H loop structure for VP1, with the structural features previously described by Logan et al. (1993) and Verdaguer et al. (1995, 1998).
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2. Unusual variants and the compromise between receptor recognition and antibody escape RNA viruses adapt by producing many mutants (in the order of one mutation in each molecule of progeny RNA copied from a template) and expanding those mutants which are most fit in the particular environment in which replication takes place (recent review in Domingo and Holland, 1997). Antigenic site A of FMDV constitutes an excellent model system to explore with molecular detail how a virus uses the same site for two seemingly contradictory purposes. One is receptor recognition, a function expected to dictate constancy of the viral sequences involved. The other is escape from antibodies which dictates variation. Part of the problem posed by these conflicting demands was solved by postulating that receptor recognition sites on the virus would be hidden from immune attack, and that they would be physically separated from the sites recognized by antibodies. However, the work with FMDV (Verdaguer et al., 1995, 1998), poliovirus (Harber et al., 1995) and rhinovirus (Smith et al., 1996) has documented that residues belonging to antigenic sites may be also involved in receptor recognition. In the case of FMDV the critical Arg-Gly-Asp triplet is involved both in integrin recognition and antibody binding. This multifunctionality should not be surprising since RNA genomes have evolved a number of molecular designs to accumulate multiple information in a limited genomic region: overlapping open-reading frames, ambisense RNA, multifunctional proteins, among other devices. Yet in all cases mutant generation and potential for rapid variation are successfully exploited for adaptation of RNA viruses (Domingo et al., 1993, 1996). In the specific case provided by site A of FMDV the solution to this riddle is being provided by new observations on receptor usage and, probably related to the latter, evidence of dispensability of residues once thought to be strictly required for cell recognition. A key observation for FMDV, and recently extended to other viruses, was that cell culture adaptation paralleled the ability of the virus to bind hep-
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arin, and to use binding to heparan sulfate as a step necessary for infection (Jackson et al., 1996; Sa-Carvalho et al., 1997). This cell tropism alteration involves acquisition by mutation of positively charged amino acids at the capsid surface, although the location of such critical residues appears to vary for different FMDV strains (Baranowski et al., 1998). It is not yet entirely clear whether heparan sulfate can act as a receptor on its own, or as a coreceptor, or simply as a first landing site as a step towards internalization by a bona fide receptor, an integrin or other. In the course of studies aimed at identifying variations in the FMDV C-S8c1 capsid that could be associated with gains in relative fitness of the virus in BHK-21 cells, we isolated a number of mutants resistant to MAb SD6. None out of 82 escape mutants of FMDV C-S8c1 included variations at the RGD triplet while, surprisingly, five out of 31 escape mutants of FMDV C-S8c1 passaged one hundred times in BHK-21 cells had substitutions at the RGD (Martı´nez et al., 1997). One of the mutants included an RGG (instead of RGD) triplet, and a number of additional capsid replacements, with acquisition of positively charged amino acids on its surface. This virus, as well as other mutants of the same passage series, acquired weak binding for heparin— a binding which is not observed with the parental C-S8c1 FMDV—and the ability to infect Chinese hamster ovary (CHO) cells. However, mutant CHO cells deficient in heparan sulfate could also be infected (Baranowski et al., 1998). This rather puzzling observation is now being investigated further. Recent experiments have documented a considerable genetic stability of the RGG mutant in cell culture. Also, this mutant can be effectively neutralized by polyclonal antibodies directed to site A of C-S8c1 (Ruiz-Jarabo et al., 1999). Thus the RGD triplet can be dispensable for efficient infection by FMDV of cells in culture. The studies of Mateu et al. (1996) and of Verdaguer et al. (1998) allow a comparison of the effects of amino acid replacements on a synthetic peptide representing antigenic site A on inhibition of FMDV infectivity and binding to MAbs.
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Although some residues have a different involvement in cell recognition and antibody binding, Gly-142 and Asp-143 of the RGD triplet, and also the neighboring residues Leu-144 and Leu147 play critical roles in the two types of interactions. Therefore, it was unexpected that a viable virus with substitution Asp-143 Gly would be readily obtained (Martı´nez et al., 1997). It is not known whether this mutant is still able to recognize an integrin receptor or it uses alternative receptors for entry into the cell. Evolution of FMDV has shaped site A in a way that it has sufficient tolerance to replacements so as to escape from neutralizing antibodies and still be able to perform the essential receptor recognition and cell entry functions (Domingo et al., 1996). The repeated finding of the same loop structure in a variety of complexes involving different antibodies and peptide antigens (Verdaguer et al., 1995, 1998, 1999; Ferna´ndez et al., manuscript in preparation) suggests that a considerable number of replacements may cause only subtle structural alterations while preserving the overall quasi-cyclical shape of the loop. Furthermore, the recent observations summarized in the present report suggest that loop variations may not only be permissive for integrin recognition but may actually be irrelevant in some cases, in view of the potential use of alternative receptors. This important question is being actively investigated by several groups.
‘down’ position found in reduced FMDV O1 BFS (Logan et al., 1993; Hewat et al. 1997; Verdaguer et al., 1999). These comparisons suggest that the loop may have a hinge movement (Parry et al., 1990) on the virion surface, perhaps contributing to its immunodominance with alternative modes of interaction with antibodies. Both the cryo-electron microscopy and X-ray crystallographic reconstructions, together with biochemical studies of neutralization by the MAbs and their Fab moieties, suggest that the antibodies neutralize by monovalent binding to the loop without aggregation of virions (Verda-
3. Current view of FMDV antigenic site A and neutralization mechanism The structures of peptide – antibody complexes (Verdaguer et al., 1995, 1998) were combined with cryo-electron microscopy studies of the same Fab molecules bound to C-S8c1 particles (Hewat et al., 1997; Verdaguer et al., 1999). This has allowed the positioning of the loop on the virion surface (Fig. 1). Interestingly the present evidence suggests that the position of the loop relative to reference residues on the capsid surface is different when it is bound to MAb SD6 than when it is bound to MAb 4C4 (Verdaguer et al., 1999). Both modeled positions are not coincident with the
Fig. 1. Ribbon diagram of a biological protomer of FMDV C-S8c1. The capsid proteins VP1, VP2 and VP3 are represented in light grey. The position of the G-H loop of VP1 was derived from cryo-electron microscopy reconstructed models of complexes between FMDV C-S8c1 and the Fab fragment of either MAb SD6 or 4C4 (Hewat et al., 1997; Verdaguer et al., 1999). The position of the loop in the SD6 and 4C4 complexes is shown in dark and medium grey, respectively. For comparison, the disposition of the loop determined in the crystallographic structure of the reduced FMDV O1BFS (Logan et al., 1993) is depicted in white. The side-chains of arginine and aspartic acid of the essential Arg-Gly-Asp triplet are shown.
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guer et al., 1997, 1998). Thus the current model is that the family of site A-specific MAbs that we have used to define the epitopes of site A (reviewed in Mateu, 1995) probably neutralize by binding to the loop and by interfering with receptor recognition. Many interesting questions remain. In some multiply passaged FMDV populations, the requirement of an RGD has been relaxed to the point that multiple variants with unusual substitutions at RGD and neighboring residues have been isolated among biological clones of the quasispecies (Ruiz-Jarabo et al., 1999). It appears as if this antigenic site could undergo a rather unrestricted diversification. Implications for FMDV pathogenesis and disease control are obvious. Would the loop maintain a quasi-circular shape (Logan et al., 1993; Verdaguer et al., 1999) when it is modified by multiple amino acid substitutions? What is the antigenic repertoire that should be incorporated in a synthetic vaccine formulation? These, and some related questions, are now under study, not only for academic reasons, but also because a broad repertoire of antibodies able to bind the loop in different manners may help in avoiding selection of FMDV-escape mutants. Thus, cocktails of different versions of antigenic site A peptides, designed according to the results of neutralization of infectivity in cell culture, and according also with the repertoire of escape mutants seen in vivo (Taboga et al., 1997), may help in the design of synthetic vaccines.
Acknowledgements We are indebted to D. Stuart, A. King, E. Hewat and their colleagues for important contributions to structural studies with FMDV, and to E. Brocchi for the generous supply of MAb 4C4. Work in Madrid was supported by grants from DGES PM97-0060-C02-01 and Fundacio´n Ramo´n Areces. Work at CID in Barcelona was supported by grant PB95-0218 from DGICYT. Work at Universitat de Barcelona was supported by grant DGICYT PB94-00845 and by Centre de Refere`ncia de Biotecnologia de la Generalitat de Catalunya. E.B. is a postdoctoral fellow from M.E.C.
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