Instituto Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi-Girona 18-26, 08034 Barcelona, Spain1
Centro de Biología Molecular Severo Ochoa', Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain2
Departament de Química Orgánica, Universitat de Barcelona, 08028 Barcelona, Spain3
Author for correspondence: Nuria Verdaguer. Fax +34 93 2045904. e-mail nvmcri{at}cid.csic.es
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Abstract |
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Introduction |
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Neutralizing antibodies are important determinants of protection against FMD and other picornavirus diseases (Mateu, 1995 ; McCullough et al., 1992
; Misbah et al., 1992
). An understanding of the types of interactions between antibodies and viruses that lead to virus neutralization is essential for vaccine design. One of the major antigenic sites of FMDV is located in the GH loop of capsid protein VP1 (Bittle et al., 1982
; Pfaff et al., 1982
; Strohmaier et al., 1982
). This loop is disordered on the surface of FMDV particles (Acharya et al., 1989
; Curry et al., 1996
; Lea et al., 1994
, 1995
; Logan et al., 1993
). For FMDV of serotype C, this antigenic site has been termed site A, and it behaves as an independent unit, with very limited influence of other capsid residues regarding the interaction of site A with antibodies (Hewat et al., 1997
; Lea et al., 1994
; Mateu, 1995
; Verdaguer et al., 1999
). The behaviour of this antigenic loop in FMDV particles in its interaction with antibodies can be mimicked faithfully with synthetic peptides that represent the relevant amino acid sequences found in authentic virus (Clarke et al., 1983
; Mateu et al., 1989
, 1990
; Mateu, 1995
; Rowlands et al., 1983
). Interestingly, antigenic site A includes a highly conserved ArgGlyAsp (RGD) triplet that serves as the recognition site of an integrin receptor (Berinstein et al., 1995
; Fox et al., 1989
; Hernández et al., 1996
; Jackson et al., 1997
; Mason et al., 1994
; Neff et al., 1998
).
The structure of a synthetic peptide representing antigenic site A of FMDV C-S8c1 [a biological clone of natural isolate C-Sta Pau Sp70 (Sobrino et al., 1983 ), a virus representative of the European subtype C1 FMDVs] in a complex with the Fab of neutralizing monoclonal antibodies (MAbs) raised against the virus has been studied by X-ray crystallography (Verdaguer et al., 1995
, 1996
, 1998
). The synthetic peptide spanned positions 136150 of capsid protein VP1 (peptide A15, Table 1
). Two complexes were studied, involving MAbs SD6 (Verdaguer et al., 1995
, 1996
) and 4C4 (Verdaguer et al., 1998
). The two neutralizing MAbs were raised against two different FMDV type C isolates (Mateu et al., 1990
): SD6 against C-S8c1 and 4C4 against C1-Brescia, a virus that differs from C-S8c1 at two positions (140 and 149) within site A (Table 1
). In the two complexes, the peptide antigen acquired a very similar quasi-circular conformation and the RGD motif participated directly in the interaction with residues of the complementarity-determining regions (CDRs) of the antibodies (Verdaguer et al., 1998
). The RGD triplet appeared in an open turn conformation, very similar to that of reduced FMDV particles of serotype O (Logan et al., 1993
), and also similar to the conformation in RGD-containing integrin ligands (Pfaff, 1997
). Remarkably, the Gly-142 and Asp-143 residues of the RGD triplet appear to be critical for interaction both with an integrin receptor and with neutralizing antibodies directed to site A (Mateu et al., 1996
; Verdaguer et al., 1995
). Both structural and biochemical evidence suggest that MAbs SD6 and 4C4 neutralize by monovalent binding to antigenic site A (Verdaguer et al., 1997
, 1998
; review in Domingo et al., 1999
).
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Methods |
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Crystallization and data collection.
Crystals of the complex between the Fab fragment of the MAb 4C4 and the variant peptide A15-CS30 (Table 1) were obtained by the hanging drop-vapour diffusion technique and successive micro- and macroseedings (Stura & Wilson, 1992
). Typically small, twined needles, obtained with 18% PEG 4K, were used for microseeding, which produced larger needles at 16% PEG 4K. Finally, these larger needles were used for macroseedings in 2 µl droplets containing 7 mg/ml Fab, 1·8 mg/ml peptide, 6·5% PEG 4K, 0·2 M LiCl with 50 mM TrisHCl (pH 9), equilibrated against a reservoir containing 13% PEG 4K equally buffered at room temperature. Crystals were orthorhombic, space group P212121 with unit cell parameters a=48·2
, b=69·3
and c=146·5
, containing one molecule of the complex per asymmetric unit, which corresponds to a solvent content volume of 49%. A data set was collected, at 100 K with 20% glycerol as a cryoprotectant, by using a MarReseach imaging plate on a Rigaku rotating anode. Intensities were evaluated and scaled internally with programs Denzo and Scalepack, respectively (Otwinowski & Minor, 1996
). Data were 93% complete at 2·3
resolution, with an internal agreement factor (Rsymm) of 8·5% (Table 2
).
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Molecular dynamics (MD) simulation.
Three sets of MD simulations, corresponding to the variable module (Fv) of the 4C4 Fab interacting with peptides A15-C-S8c1, A15-C-S8c1/LV and A15-C-S30 (Table 1), were performed by using the GROMOS96 package with its standard protein and water force fields (van Gunsteren et al., 1996
). Crystallographic coordinates of peptides A15-C-S8c1 and A15-C-S30 (residues 137148) were used as starting models in the corresponding simulations. Thr-148, not visible in the electron density maps of the A15-C-S8c1 complex, was added to avoid end effects in the critical Leu-147 residue, by using the information available in the A15-C-S30 structure. Starting coordinates for the A15-C-S8c1/LV peptide were then derived, with the graphic program TURBO (Rousel & Cambillau, 1989
), by replacing Leu-147 with valine. Crystallographic water molecules determined in the present work for the complex of 4C4 Fab with the A15-C-S30 peptide were not included in the simulations.
In every simulation, the structure of the corresponding complex was placed initially at the centre of a truncated octahedron, the dimensions of which were chosen such that the minimum distance of any protein atom from the closest wall was 7 . The edge lengths of the corresponding cubic boxes were about 73
. Systems were treated as immersed into an equilibrium configuration of bulk simple point charge (SPC) water (Berendsen et al., 1986
). Water molecules outside the box or with a distance to a solute atom of less than 2·3
were removed. The numbers of water molecules considered in the three simulations were 5271, 5272 and 5264, respectively. To relax strong waterwater and waterprotein non-bonded interactions, steepest-descent energy minimization was performed until stabilization was reached. After that, counter-ions were added to neutralize charged protein sites, with a subsequent energy minimization. Simulations were performed at constant volume and temperature (300 K) with periodic boundary conditions and an integration step of 2 fs. Temperature was kept constant by weak coupling to an external bath (Berendsen et al., 1984
, 1986
). Bond lengths were constrained to equilibrium values by using the SHAKE algorithm (Rickaert et al., 1977
). Equilibrations were achieved within 50 ps and, afterwards, the three systems were simulated for a total time of 300 ps with the total potential energy remaining essentially constant. Only amino acids from the peptides and all water molecules were allowed to move during calculations. Analysis were performed, mainly with programs contained in the GROMOS96 package, using the structures generated every 0·1 ps during the interval spanning from equilibration until the end (from about 50 to 350 ps).
Coordinates.
Coordinates have been deposited in the Brookhaven protein database under accession number 1EJO.
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Results |
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Comparisons of the site A variant peptides A15-CS30 and A15-CS8c1
The root-mean-square deviation (RMSD) between the main-chain peptide atoms in the A15-C-S30 and A15-C-S8c1 Fab 4C4 complexes is only 0·4 for the common residues 137147. The Arg-141 side chain has different dispositions in the two peptides (Fig. 1C
), although the electron density corresponding to this residue was weak in both complexes (Fig. 1A
; see also Verdaguer et al., 1995
, 1998
). The replaced residues, Thr-138 and Val-147, show the largest main-chain deviations of 0·7 and 1·0
, respectively. Despite the high structural similarity between the two peptides A15-C-S30 and A15-C-S8c1, differences in the main-chain conformational angles are important, particularly around the RGD motif (Fig. 2
). Thus, in the A15-C-S30 peptide, residues Thr-140 and Arg-141 are situated in the Ramachandran region corresponding to
-helices while, in the A15-C-S8c1 peptide, Ala-140 and Arg-141 are in the regions of
-strands and left-handed helices, respectively. A third situation is found in the structure reported for the GH loop of the reduced FMDV serotype O1 (Lea et al., 1993
, 1995
), where the corresponding residues Leu-144 and Arg-145 are both found in the Ramachandran region corresponding to
-strands. The flexibility of Gly-142 allows compensatory main-chain torsional angles that result in the overall structural similarity of the three peptides. It is important to notice the presence, in the A15-CS30 structure, of one water molecule (named w8 in Fig. 1
) that is hydrogen-bonded with the side chain of Thr-138 and the main-chain oxygen atoms of residues Leu-144 and Leu-147. This water molecule cannot be present, for steric reasons, in the A15-C-S8c1 structure, where the bulkier Leu-147 side chain fills the available space (Fig. 3
).
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Discussion |
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In the structure of the 4C4 Fab complexed with the variant peptide A15-C-S30 reported here, the two amino acids Thr-138 and Val-147 that were replaced in the peptide show only minor interactions with the Fab (Table 2 and Fig. 2
). Differences in binding affinities for peptides with substitutions in those two residues should therefore be due mainly to free-energy differences when adopting a correct' site A-like loop structure, the one recognized by antibodies or by the receptor (Fig. 5
). The reduced stability of that conformation in the peptide with the single substitution Leu-147
Val would then explain the reduced affinity observed for the antibody. Instead, the double-substituted peptide could recover binding affinity by re-stabilizing the correct' conformation with the additional water molecule (w8) that bridges the chain hydroxyl group of the replaced Thr-138 with the main-chain oxygen atoms of residues Leu-144 and Leu-147 (Table 3
and Fig. 3C
). In the MD simulation of the A15-C-S30 variant peptide, a water molecule with a low RMSD is placed in almost exactly the same location as the one found in the crystal structure (Fig. 4B
). These structural results provide a rationale for how a general change in specificity to a large diversity of antibodies can be achieved by a destabilization of the original antigen structure. In those cases, further substitutions can restore the affinity for the antibodies by increasing the stability of the original antigen conformation, even when none of the residues substituted participates in direct antigenantibody interactions (Freire, 1999
).
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Acknowledgments |
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References |
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Bachrach, H. L. (1968). Foot and mouth disease virus.Annual Review of Microbiology 22, 201-244.[Medline]
Beck, E. & Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. Journal of Virology 61, 1621-1629.[Medline]
Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. (1984). Molecular dynamics with coupling to an external bath.Journal of Chemical Physics 81, 3684-3690.
Berendsen, H. J., Van Gunsteren, W. F., Zwinderman, H. R. & Geurtsen, R. G. (1986). Simulations of proteins in water.Annals of the New York Academy of Sciences 482, 269-286.[Abstract]
Berinstein, A., Roivainen, M., Hovi, T., Mason, P. W. & Baxt, B. (1995). Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.Journal of Virology 69, 2664-2666.[Abstract]
Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M., Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. & Brown, F. (1982). Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence.Nature 298, 30-33.[Medline]
Brown, F. (1994). Foot-and-mouth disease. In Synthetic Vaccines, pp. 416-432. Edited by B. H. Nicholson. Oxford: Blackwell Scientific.
Brünger, A. T. (1992). XPLOR manual, version 3.0. Yale University, New Haven, CT, USA.
Carreño, C., Roig, X., Cairo, J., Camarero, J., Mateu, M. G., Domingo, E., Giralt, E. & Andreu, D. (1992). Studies on antigenic variability of C strains of foot-and-mouth disease virus by means of synthetic peptides and monoclonal antibodies.International Journal of Peptide and Protein Research 39, 41-47.[Medline]
Clarke, B. E., Carroll, A. R., Rowlands, D. J., Nicholson, B. H., Houghten, R. A., Lerner, R. A. & Brown, F. (1983). Synthetic peptides mimic subtype specificity of foot-and-mouth disease virus.FEBS Letters 157, 261-264.[Medline]
Curry, S., Fry, E., Blakemore, W., Abu-Ghazaleh, R., Jackson, T., King, A., Lea, S., Newman, J., Rowlands, D. & Stuart, D. (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]
Domingo, E. & Holland, J. J. (1992). Complications of RNA heterogeneity for the engineering of virus vaccines and antiviral agents. In Genetic Engineering, Principles and Methods, pp. 13-32. Edited by J. K. Setlow. New York: Plenum.
Domingo, E., Mateu, M. G., Martínez, M. A., Dopazo, J., Moya, A. & Sobrino, F. (1990). Genetic variability and antigenic diversity of foot-and-mouth disease virus. In Applied Virology Research, pp. 233-266. Edited by E. Kurkstak, R. G. Marusyk, S. A. Murphy & M. H. V. Van Regenmortel. New York: Plenum.
Domingo, E., Verdaguer, N., Ochoa, W. F., Ruiz-Jarabo, C. M., Sevilla, N., Baranowski, E., Mateu, M. G. & Fita, I. (1999). Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus. Virus Research 62, 169-175.[Medline]
Fox, G., Parry, N. R., Barnett, P. V., McGinn, B., Rowlands, D. J. & Brown, F. (1989). The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). Journal of General Virology 70, 625-637.[Abstract]
Freire, E. (1999). The propagation of binding interactions to remote sites in proteins: analysis of the binding of the monoclonal antibody D1.3 to lysozyme. Proceedings of the National Academy of Sciences, USA 96, 10118-10122.
Hernández, J., Valero, M. L., Andreu, D., Domingo, E. & Mateu, M. G. (1996). Antibody and host cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif: a structural interpretation. Journal of General Virology 77, 257-264.[Abstract]
Hewat, E. A., Verdaguer, N., Fita, I., Blakemore, W., Brookes, S., King, A., Newman, J., Domingo, E., Mateu, M. G. & Stuart, D. I. (1997). Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop. EMBO Journal 16, 1492-1500.
Jackson, T., Sharma, A., Abu-Ghazaleh, R., Blakemore, W. E., Ellard, F. M., Simmons, D. L., Newman, J. W., Stuart, D. I. & King, A. M. (1997). Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin v
3 in vitro. Journal of Virology 71, 8357-8361.[Abstract]
Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallographica A47, 110-119.[Medline]
Lea, S., Hernández, J., Blakemore, W., Brocchi, E., Curry, S., Domingo, E., Fry, E., Abu-Ghazaleh, R., King, A., Newman, J., Stuart, D. & Mateu, M. G. (1993). Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 362, 566-568.[Medline]
Lea, S., Hernández, J., Blakemore, W., Brocchi, E., Curry, S., Domingo, E., Fry, E., Abu-Ghazaleh, R., King, A., Newman, J., Stuart, D. & Mateu, M. G. (1994). The structure and antigenicity of a type C foot-and-mouth disease virus. Structure 2, 123-139.[Medline]
Lea, S., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Fry, E., Jackson, T., King, A., Logan, D., Newman, J. & Stuart, D. (1995). Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67. Structure 3, 571-580.[Medline]
Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis, R., Newman, J., Parry, N., Rowlands, D., Stuart, D. & Fry, E. (1993). Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 362, 566-568.[Medline]
McCullough, K. C., De Simone, F., Brocchi, E., Capucci, L., Crowther, J. R. & Kihm, U. (1992). Protective immune response against foot-and-mouth disease. Journal of Virology 66, 1835-1840.[Abstract]
Martínez, M. A., Hernández, J., Piccone, M. E., Palma, E. L., Domingo, E., Knowles, N. & Mateu, M. G. (1991). Two mechanisms of antigenic diversification of foot-and-mouth disease virus. Virology 184, 695-706.[Medline]
Martínez, M. A., Verdaguer, N., Mateu, M. G. & Domingo, E. (1997). Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus . Proceedings of the National Academy of Sciences, USA 94, 6798-6802.
Mason, P. W., Rieder, E. & Baxt, B. (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. Proceedings of the National Academy of Sciences, USA 91, 1932-1936.[Abstract]
Mateu, M. G. (1995). Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus Research 38, 1-24.[Medline]
Mateu, M. G., Rocha, E., Vicente, O., Vayreda, F., Navalpotro, C., Andreu, D., Pedroso, E., Giralt, E., Enjuanes, L. & Domingo, E. (1987). Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease. Virus Research 8, 261-274.[Medline]
Mateu, M. G., Da Silva, J. L., Rocha, E., De Brum, D. L., Alonso, A., Enjuanes, L., Domingo, E. & Barahona, H. (1988). Extensive antigenic heterogeneity of foot-and-mouth disease virus of serotype C. Virology 167, 113-124.[Medline]
Mateu, M. G., Martínez, M. A., Rocha, E., Andreu, D., Parejo, J., Giralt, E., Sobrino, F. & Domingo, E. (1989). Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus. Proceedings of the National Academy of Sciences, USA 86, 5883-5887.[Abstract]
Mateu, M. G., Martínez, M. A., Capucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E. & Domingo, E. (1990). A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C. Journal of General Virology 71, 629-637.[Abstract]
Mateu, M. G., Andreu, D., Carreño, C., Roig, X., Cairó, J. J., Camarero, J. A., Giralt, E. & Domingo, E. (1992). Non-additive effects of multiple amino acid substitutions on antigenantibody recognition. European Journal of Immunology 22, 1385-1389.[Medline]
Mateu, M. G., Valero, M. L., Andreu, D. & Domingo, E. (1996). Systematic replacement of amino acid residues within an Arg-Gly-Asp-containing loop of foot-and-mouth disease virus and effect on cell recognition. Journal of Biological Chemistry 271, 12814-12819.
Misbah, S. A., Spickett, G. P., Ryba, P. C., Hockaday, J. M., Kroll, J. S., Sherwood, C., Kurtz, J. B., Moxon, E. R. & Chapel, H. M. (1992). Chronic enteroviral meningoencephalitis in agammaglobulinemia: case report and literature review. Journal of Clinical Immunology 12, 266-270.[Medline]
Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallographica A50, 157-163.
Neff, S., Sá-Carvalho, D., Rieder, E., Mason, P. W., Blystone, S. D., Brown, E. J. & Baxt, B. (1998). Foot-and-mouth disease virus virulent for cattle utilizes the integrin v
3 as its receptor. Journal of Virology 72, 3587-3594.
Novella, I. S., Borrego, B., Mateu, M. G., Domingo, E., Giralt, E. & Andreu, D. (1993). Use of substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot-and-mouth disease virus. FEBS Letters 330, 253-259.[Medline]
Otwinowski, Z. & Minor, W. (1996). Processing of X-ray diffraction data collected in an oscillation mode. Methods in Enzymology 35, 307-326.
Parry, N., Fox, G., Rowlands, D., Brown, F., Fry, E., Acharya, R., Logan, D. & Stuart, D. (1990). Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus. Nature 347, 569-572.[Medline]
Pereira, H. G. (1977). Subtyping foot-and-mouth disease virus. Developments in Biological Standardization 35, 167-174.
Pereira, H. G. (1981). Foot-and-mouth disease virus. In Virus Diseases of Food Animals, pp. 333-363. Edited by R. P. G. Gibbs. New York: Academic Press.
Pfaff, E. (1997). Recognition sites of RGD-dependent integrins. In Integrin Ligand Interactions, pp. 101-121. Edited by J. A. Eble & K. Kühn. Austin, TX: R. G. Landes.
Pfaff, E., Mussgay, M., Bohm, H. O., Schulz, G. E. & Schaller, H. (1982). Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus. EMBO Journal 1, 869-874.[Medline]
Rickaert, J. P., Ciccotti, G. & Berendsen, H. J. C. (1977). Numerical integration of cartesian equations of motions of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics 23, 327-341.
Rousel, A. & Cambillau, C. (1989). TURBO-Frodo. In Silicon Graphics Geometry Partners Directory, pp. 7779. Mountain View, CA: Silicon Graphics.
Rowlands, D. J., Clarke, B. E., Carroll, A. R., Brown, F., Nicholson, B. H., Bittle, J. L., Houghten, R. A. & Lerner, R. A. (1983). Chemical basis of antigenic variation in foot-and-mouth disease virus. Nature 306, 694-697.[Medline]
Rueckert, R. R. (1996). Picornaviridae: the viruses and their replication. In Fields Virology, pp. 609-654. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Sobrino, F., Dávila, M., Ortín, J. & Domingo, E. (1983). Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture. Virology 128, 310-318.[Medline]
Strohmaier, K., Franze, R. & Adam, K.-H. (1982). Location and characterization of the antigenic portion of the FMDV immunizing protein. Journal of General Virology 59, 295-306.[Abstract]
Stura, E. A. & Wilson, I. A. (1992). Seeding techniques. In Crystallization of Nucleic Acids and Proteins. A Practical Approach, pp. 99-126. Edited by A. Ducruix & R. Giege. Oxford: IRL Press.
Taboga, O., Tami, C., Carrillo, E., Núñez, J. I., Rodríguez, A., Saíz, J. C., Blanco, E., Valero, M. L., Roig, X., Camarero, J. A., Andreu, D., Mateu, M. G., Giralt, E., Domingo, E., Sobrino, F. & Palma, E. L. (1997). A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants. Journal of Virology 71, 2606-2614.[Abstract]
Timoney, J. F., Gillespie, J. H., Scott, F. N. & Barlough, J. E. (1992). Foot-and-mouth disease. In Hagan and Bruners Microbiology and Infectious Diseases of Domestic Animals, pp. 647667. Ithaca, NY: Cornell University Press.
van Gunsteren, W. F., Billeter, S. R., Eising, A. A., Hünenberg, P. H., Krüger, P., Mark, A. E., Scott, W. R. P. & Tironi, I. G. (1996). Biomolecular simulation. In GROMOS96 Manual and User Guide. Zürich: Biomolecular Software.
Verdaguer, N., Mateu, M. G., Andreu, D., Giralt, E., Domingo, E. & Fita, I. (1995). Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO Journal 14, 1690-1696.[Abstract]
Verdaguer, N., Mateu, M. G., Bravo, J., Domingo, E. & Fita, I. (1996). Induced pocket to accommodate the cell attachment Arg-Gly-Asp motif in a neutralizing antibody against foot-and-mouth disease virus. Journal of Molecular Biology 256, 364-376.[Medline]
Verdaguer, N., Fita, I., Domingo, E. & Mateu, M. G. (1997). Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding. Journal of Virology 71, 9813-9816.[Abstract]
Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E., Mateu, M. G. & Fita, I. (1998). A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. Journal of Virology 72, 739-748.
Verdaguer, N., Schoehn, G., Ochoa, W. F., Fita, I., Brookes, S., King, A., Domingo, E., Mateu, M. G., Stuart, D. & Hewat, E. A. (1999). Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralizing antibody: structure and neutralization. Virology 255, 260-268.[Medline]
Villaverde, A., Martínez, M. A., Sobrino, F., Dopazo, J., Moya, A. & Domingo, E. (1991). Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock? Gene 103, 147-153.[Medline]
Received 2 December 1999;
accepted 22 February 2000.