Exploring nucleic acid condensation and release from individual parvovirus particles with different physicochemical cues.

In the infection cycle, viruses release their genome in the host cell during uncoating. Here we use a variety of physicochemical procedures to induce and monitor the in vitro uncoating of ssDNA from individual Minute Virus of Mice (MVM) particles. Our experiments revealed two pathways of genome release: i) filamentous ssDNA appearing around intact virus particles when using gradual mechanical fatigue and heating at moderate temperature (50 °C). ii) thick structures of condensed ssDNA appearing when the virus particle is disrupted by mechanical nanoindentations, denaturing agent guanidinium chloride and high temperature (70 °C). We propose that in the case of filamentous ssDNA, when the capsid integrity is conserved, the genome is externalized through one channel of the capsid pores. However, the disruption of virus particles revealed a native structure of condensed genome. The mechanical analysis of intact particles after DNA strands ejection confirm the stabilization role of ssDNA in MVM.

Quantitative nanoscale electrostatics of viruses.

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed ϕ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.

The capillarity of nanometric water menisci confined inside closed-geometry viral cages.

We present an investigation of water menisci confined in closed geometries by studying the structural effects of their capillary forces on viruses during the final stage of desiccation. We used individual particles of the bacteriophage phi29 and the minute virus of mice. In both cases the genomic DNA was ejected from the capsid. However, although the structural integrity of the minute virus of mice was essentially preserved, the phi29 capsid underwent a wall-to-wall collapse. We provide evidence that the capillary forces of water confined inside the viruses are mainly responsible for these effects. Moreover, by performing theoretical simulations with a lattice gas model, we found that some structural differences between these 2 viruses may be crucial to explain the different ways in which they are affected by water menisci forces confined at the nanoscale.

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 approximately 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.

Hydrogen exchange of the tetramerization domain of the human tumour suppressor p53 probed by denaturants and temperature.

We have analysed the hydrogen/deuterium exchange of the tetramerization domain of human tumour suppressor p53 under mild chemical denaturation conditions, and at different temperatures. Exchange behaviour has been measured for 16 amide protons in the chemical-denaturation studies and for seven protons in the temperature-denaturation studies. The exchange rates are in the range observed for other proteins with similar elements of secondary structure. The slowest-exchange core includes contributions from residues in the alpha helix and the beta sheet. However, only some of the slowest-exchanging protons correspond to residues involved in native interactions in the transient intermediate detected during the folding of this domain. The guanidinium-chloride denaturation curves of all residues seem to merge together, although they are well below the main isotherm of global unfolding. Thus, there is no evidence for several subglobal unfolding units. The activation parameters obtained from the temperature-denaturation experiments are similar to those obtained for monomeric proteins, and well below the global unfolding enthalpy obtained by circular dichroism measurements. Thus, the exchange studies at different denaturant concentrations and temperatures indicate that no particular folding intermediate is populated under those conditions.

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity and flexibility.

Antigenic site A of foot-and-mouth disease virus (serotype C) has been reproduced by means of cyclic versions of peptide A15, YTASARGDLAHLTTT, corresponding to residues 136-150 of envelope protein VP1. A structural basis for the design of the cyclic peptides is provided by crystallographic data from complexes between the Fab fragments of anti-site A monoclonal antibodies and A15, in which the bound peptide is folded into a quasi-cyclic pattern. Head-to-tail cyclizations of A15 do not provide peptides of superior antigenicity. Internal disulfide cyclization, however, leads to analogs which are recognized as one to two orders of magnitude better than linear A15 in both ELISA and biosensor experiments. CD and NMR studies show that the best antigen, CTASARGDLAHLTT-Ahx-C (disulfide), is very insensitive to environment-induced conformational change, suggesting that cyclization helps to stabilize a bioactive-like structure.

Protein folding transition states: elicitation of Hammond effects by 2,2,2-trifluoroethanol.

Adaptation of the techniques of classical physical-organic chemistry to the study of protein folding has led to our current detailed understanding of the transition states. Here, we have applied a series of structure--activity relationships to analyse the effects on protein folding transition states of 2,2,2-trifluoroethanol (TFE), a reagent that is usually assumed to act by stabilising secondary structure. The folding and unfolding of the highly alpha-helical tetramerisation domain of p53 provides a useful paradigm for analysing its effects on kinetics: The first step of its folding consists of an association reaction with little, if any, formation of secondary structure in the transition state; and the final step of the folding reaction involves just the formation of bonds at subunit interfaces, with the alpha-helical structure being completely formed. We have systematically measured the effects of TFE on two sets of structure--activity relationships. The first is for Phi values, which measure the degree of non-covalent bond formation at nearly every position in the transition state. The second is for relative effects of the denaturant, guanidinium chloride, on kinetics and equilibria, which measure the gross position of the transition state on the reaction co-ordinate. We find that TFE modulated the kinetics by a variety of effects other than that on secondary structure. In particular, there were Hammond effects, movement of the position of the transition state along the reaction co-ordinate, which either significantly speeded up or slowed down protein unfolding, depending on the particular mutant examined. The gross effects of TFE on protein folding kinetics are thus not a reliable guide to the structures of transition states.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

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.

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization.

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.

Mechanism of folding and assembly of a small tetrameric protein domain from tumor suppressor p53.

We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protein p53. Structures of transition states were determined from phi-values for 25 mutations, including leucine to norvaline, and the analysis encompassed nearly every residue in the domain. Denatured monomers fold and dimerize, through a transition state with little native structure, to form a transient, highly structured dimeric intermediate. The intermediate dimerizes, through a native-like transition state with the primary dimers fully folded but with interdimer interactions only partially formed, to form the native tetramer as a 'dimer of dimers'.

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.

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.

Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus.

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet-which is also a widespread motif involved in cell-to-cell adhesion-can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature.

A cyclic disulfide peptide reproduces in solution the main structural features of a native antigenic site of foot-and-mouth disease virus.

A cyclic disulfide peptide corresponding to the G-H loop sequence 134-155 [replacement Tyr136 and Arg153 with Cys] of the capsid protein VP1 of foot-and-mouth disease virus (FMDV) isolate C-S8c1 was examined by proton 2D-NMR spectroscopy in water and in 25% HFIP/water. In water, NMR data supported the presence of a non-canonical turn in the central, conserved cell adhesion RGD motif and suggested the presence of a nascent helix in the C-terminal part, stabilized and slightly extended upon addition of 25% HFIP, a secondary structure stabilizing cosolvent. The formation of the C-terminal helix was evidenced by combined analysis of NOE connectivities, H alpha chemical shifts, 3JNH-H alpha coupling constants and amide temperature coefficients. Surprisingly, these global structural features of the cyclic peptide in solution show similarities to previous X-ray structure analysis of (a) a shortened linear peptide complexed with a antivirus antibody and (b) the G-H loop represented on the chemical reduced viral surface of a different serotype. Thus, even in entirely different biological environments the cyclic peptide reflect similar structural features, reinforcing the concept that this viral loop behaves as an independent structural and functional unit.

A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants.

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.

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.

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 A resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.