Mauricio G. Mateu

Antibody recognition of picornaviruses and escape from neutralization: a structural view

Escape of picornaviruses from neutralization by monoclonal antibodies is mediated by substitutions of very few, defined amino acid residues of the capsid, generally located on the tip of some surface-exposed loops. Substitutions at the same positions are possibly of major relevance to antigenic variation of picornaviruses in the field. Such residues tend to cluster in discrete areas, termed antigenic sites. The structure of virus-antibody and peptide-antibody complexes, determined by cryoelectron microscopy and X-ray crystallography, combined with studies using site-directed mutagenesis, are beginning to reveal new features of picornavirus epitopes. This information complements and expands the view on picornavirus antigenicity previously provided by analyses of antibody-escape mutants. In addition to amino acids found replaced in escape mutants, other surface residues which remain invariant in spite of immune pressure also participate in contacts with the antibody molecule. Some invariant residues are even critical for the antigen-antibody interaction. Escape mutations occur at the subset of antigenically critical residues which are tolerant to change because they are not essentially involved in capsid structure or function. Restrictions to variation differ among epitopes; this may contribute to explain the different number of serotypes among picornaviruses, and the frequency at which antigenically highly divergent variants occur in the field.

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C.

Neutralizing monoclonal antibodies (nMAbs) elicited against foot-and-mouth disease virus (FMDV) of serotype C were assayed with field isolates and variant FMDVs using several immunoassays. Of a total of 36 nMAbs tested, 23 recognized capsid protein VP1 and distinguished at least 13 virion conformation-independent epitopes involved in neutralization of FMDV C. Eleven epitopes of FMDV C-S8c1 have been located in segments 138-156 or 192-209 of VP1 by quantifying the reactivity of nMAbs with synthetic peptides and with nMAb-resistant mutants of FMDV C-S8c1 carrying defined amino acid substitutions. The main antigenic site of FMDV C-S8c1 (VP1 residues 138 to 150) consists of multiple (at least 10), distinguishable, overlapping epitopes. Some amino acid replacements abolished one of the epitopes, whereas other replacements affected several epitopes in this region. The conservative substitution His(146)----Arg, found in many nMAb-resistant mutants analysed, abolished the reactivity of the virus with all nMAbs that recognized epitopes in the main antigenic site of FMDV C-S8c1. This indicates that a minimum genetic change can result in a highly amplified phenotypic effect, as regards the antigenicity of FMDV.

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.

The crystal structure of a synthetic peptide representing the major antigenic loop of foot‐and‐mouth disease virus (FMDV), complexed with the Fab fragment of a neutralizing monoclonal antibody raised against the virus, has been determined at 2.8 A resolution. The peptide shows a high degree of internal structure with a nearly cyclic conformation. The conserved Arg‐Gly‐Asp motif, involved in the viral attachment of aphtoviruses to cells, participates directly in the interaction with several complementarity determining regions of the antibody molecule. The Arg‐Gly‐Asp triplet shows the same open turn conformation found in the reduced form of FMDV of another serotype and also in integrin binding proteins. The observed interactions provide a molecular interpretation of the amino acid replacements observed to occur in mutants resistant to neutralization by this antibody. The structure also suggests a number of restrictions to variation within the epitope which are imposed to keep the Arg‐Gly‐Asp motif in its functional conformation.

DNA-mediated anisotropic mechanical reinforcement of a virus

In this work, we provide evidence of a mechanism to reinforce the strength of an icosahedral virus by using its genomic DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice are investigated by using atomic force microscopy. The stiffness of the empty capsid is found to be isotropic. Remarkably, the presence of the DNA inside the virion leads to an anisotropic reinforcement of the virus stiffness by ≈3%, 40%, and 140% along the fivefold, threefold, and twofold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid. These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the fivefold symmetry axes, suggest that the bound DNA may reinforce the overall stiffness of the viral particle without canceling the conformational changes needed for its infectivity.

Genetic variability and antigenic diversity of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an acute systemic disease of cloven-hooved animals, including cattle, swine, sheep, and goats. Despite mortality rates being generally below 5%, FMD severely decreases livestock productivity and trade. It is considered the economically most important disease of farm animals. Near two thousand million doses of vaccine are used annually to try to control FMD, which, nevertheless, is enzootic in most South American and African countries, parts of Asia, the Middle East, and the south of Europe. The causative agent, foot-and-mouth disease virus (FMDV), is an aphthovirus of the family Picornaviridae, a historically important virus as it was the first recognized viral agent (Loeffler and Frosch, 1898). In this chapter we review briefly the structure of FMDV and the organization and expression of its genome (Section II) and, in more detail, recent results on genetic variability (Section III) and antigenic diversity (Section V), reflected in several serotypes and subtypes of the virus. Such a diversity has implications for vaccine design and disease control, as discussed in Section V. Finally, we propose a model of evolution of FMDV and discuss its implications (Sections VI and VII).

Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain

The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326–353) of human p53 has been quantitated using 25 mutants with single‐residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8–9 kcal/mol of dimer) instead of tetramers. One‐third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1–5.7 kcal/mol). Strongly destabilizing (8.8– 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.

New observations on antigenic diversification of RNA viruses. Antigenic variation is not dependent on immune selection

Recent results have revealed novel features in the process of antigenic diversification of FMDV. (i) Antigenic variation is not necessarily the result of immune selection. (ii) Single, critical amino acid replacements may either have a minor effect on antigenic specificity or cause a drastic antigenic change affecting many epitopes on an antigenic site. (iii) The effect of such a critical replacement may be suppressed by additional substitutions at neighbouring sites. (iv) Antigenic diversification does not necessarily involve net accumulation of amino acid substitutions over time. We review evidence that some of these features apply also to other riboviruses and retroviruses. A model is proposed to relate antigenic variation without immune selection to the quasispecies structure of RNA virus populations.

Foot-and-Mouth Disease Virus Populations Are Quasispecies

The term “quasispecies” describes complex distributions of replicating molecules subject to mutation and competitive selection (Eigen 1971; Eigen and Schuster 1979; Eigen and Biebricher 1988). The original theoretical concept of Eigen and colleagues concerned populations of infinite numbers of individual molecules and ideal, steady-state equilibrium conditions (recent review on the theoretical concept in Eigen et al. 1989). It is clear that in spite of their large population size, RNA viruses deviate from such idealized behaviour. However, several key features of RNA viruses such as nucleotide sequence heterogeneity, generally high mutation rates, and potential for very rapid evolution are best understood in the framework of the quasispecies concept (see the chapter by Holland et al.). Viral isolates, either in their natural niche or disturbed by adaptation to growing in cell culture, consist of a multitude of viable and defective mutants termed the “mutant spectrum” of the population. During replication, each genomic distribution is dominated by one (or several) “master sequence (s),” that generally coincides with the average or consensus sequence of the population. With the levels of genetic heterogeneity for foot- and-mouth disease virus (FMDV) documented in the following paragraphs, the master sequence often represents as little as 1% or less of the population of molecules, and it may have a brief life span. Here we review the evidence for the quasispecies structure of FMDV and its biological implications, notably the antigenic diversity of this widespread pathogen.

Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease

A panel of 12 monoclonal antibodies (MAbs) raised against foot-and-mouth disease virus (FMDV) of serotype C1 (FMDV C-S8c1) and 11 MAbs raised against other FMDVs have been used to evaluate the reactivity of 14 isolates of FMDV of serotype C1 (series FMDV C-S), 12 of them from one disease episode (Spain 1979-1982). The assays used were immunoelectrotransfer blot, immunodot and neutralization of infectivity. None of the isolates could be clearly distinguished by its reactivity with 6 non-neutralizing and 2 neutralizing MAbs raised against FMDV C-S8c1. In contrast, the isolates were distinguished in two groups by a 10(2)-fold difference in their reactivity with 6 neutralizing MAbs. The reactivity of MAbs with synthetic peptides indicated that conserved and non-conserved epitopes recognised respectively by neutralizing MAbs 4G3 and SD6 are localized in the immunogenic region (amino acids 138-156) of VP1. Thus, epidemiologically related FMDVs differ in at least one epitope critical for virus neutralization.