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.

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.

Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos.

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.

Induced pocket to accommodate the cell attachment Arg-Gly-Asp motif in a neutralizing antibody against foot-and-mouth-disease virus.

The three-dimensional structure of the Fab fragment of a neutralizing monoclonal antibody (SD6) elicited against foot-and-mouth disease virus (FMDV) has been determined at 2.5 A resolution and refined to a crystallography agreement R-factor of 0.186. The structure has been compared with that of the same Fab molecule complexes with a 15 amino acid peptide (A15) representing a major antigenic site of FMDV, and determined at 2.8 A resolution. The Fab quaternary structure, defined both by the elbow angle between modules and by the relative disposition of the light and heavy domains inside the modules, remains essentially unchanged. However, the comparison shows important conformational variations in the paratope, especially in the hypervariable loops of the heavy chain. The CDR-H3 loop has a peculiar amino acid sequence (RREDGGDEGF) with a high content of charged residues. Some of these Fab residues were fully reoriented upon complex formation. The reorientation resulted not only in an alteration of shape but also in an important redistribution of charges, providing multiple points of interaction with the A15 antigen and in particular with the cell attachment Arg-Gly-Asp motif in the peptide. Thus the recognition of A15 by SD6 represents an extreme example of the induced fit mechanism in antibody interactions. The electron density maps provide evidence that in the uncomplexed Fab structure some CDR residues show, with lower occupancy, the conformations found in the complex, suggesting that the rearrangements observed can have only minor energetic requirements.

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.

Cyclic disulfide model of the major antigenic site of serotype-C foot-and-mouth disease virus. Synthetic, conformational and immunochemical studies.

A cyclic disulfide peptide representing antigenic site A of foot-and-mouth disease virus (FMDV) strain C-S8c1 (residues 134 to 155 of viral protein 1 (VP1) with Tyr136 and Arg153 replaced by cystine; TTCTASARGDLAHLTTTHACHL) was synthesized by solid phase methods. Formation of the cyclic disulfide was carried out by air oxidation of the fully deprotected and reduced bis-cysteine precursor, under high dilution conditions. The identity of the cyclic peptide was confirmed by both physical and enzymatic methods. A conformational study of the cyclic peptide and of its linear parent structure (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1 of FMDV C-S8c1) by circular dichroism in the presence of a structure-inducing solvent showed the cyclic disulfide analog to adopt lower levels of alpha-helix than its linear counterpart. In competitive ELISA assays both peptides reacted with similar affinity against a representative panel of neutralizing monoclonal antibodies directed towards antigenic site A. Thus, a high inherent flexibility of this loop may preclude a conformational restriction strong enough to alter recognition by anti-virus antibodies.

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.

Antigenic site A of foot-and-mouth disease virus (FMDV) is an exposed, mobile loop which includes a central, highly conserved Arg-Gly-Asp tripeptide (RGD, VP1 residues 141-143 in serotype C) thought to be part of the cell attachment site. We have analyzed the contribution of RGD to the interaction of site A with antibodies by incorporating selected amino acid replacements at RGD into synthetic peptides representing site A, and analyzing the reactivity of substituted peptides with site A-specific monoclonal antibodies (MAbs). Replacement of Arg-141, Gly-142 or Asp-143 by alanine resulted in the loss of one, three and five epitopes, respectively, out of seven epitopes probed. Other replacements resulted in the loss of even larger numbers of epitopes, suggesting that the amino acids of the RGD region are either directly involved in interaction with antibodies or that they exert an important influence on the interaction of surrounding residues with antibodies. Thus, we explored the ability of tandem repeats of the RGDL sequence (corresponding to FMDV C-S8c1) to evoke neutralizing antibodies in rabbits and guinea pigs. Neutralizing activity was generally low but with a broad specificity for different FMDV serotypes and variants. Significant decreases in neutralizing titers were observed with boosting, suggesting a possible suppression of those anti-peptide antibodies which may also be directed to cellular RGD sequences. The results point to an involvement of RGD in the antigenic structure of site A, and open the possibility that broadly neutralizing antibodies might be induced by tandem repeats of the critical, conserved domain.

Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes.

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.

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.

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.

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.

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.

A highly divergent antigenic site of foot-and-mouth disease virus retains its immunodominance.

The ability of a highly divergent antigenic site of foot-and-mouth disease virus (FMDV) of serotype C to elicit neutralizing antibodies has been evaluated in mice and rabbits. The viruses compared, FMDV C-S8c1 and HR, differ in a single amino acid replacement in their capsid proteins, but represent two extreme antigenic specificities of the major antigenic site A of FMDV type C. Both, studies of cross-neutralization of homologous and heterologous virus, and fractionation of site A-specific antibodies by immunoaffinity chromatography suggest a similar immunodominance of antigenic site A in FMDV C-S8c1 and variant HR. This information is relevant to the formulation of synthetic peptide vaccines that ideally should consist of mixtures of peptides representing several antigenic specificities. These cocktail formulations may be required to control diseases caused by FMDV and, generally, by highly variable RNA viruses, since single specificity peptides may trigger selection of vaccine-escape viral mutants.

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.

Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface.

A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1. We have studied the antigenicity of several recombinant E. coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme. In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A. In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus. In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity. Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers. These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site.

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.

Protein synthesis in Drosophila melanogaster embryos. Two mechanisms for guanine nucleotide exchange on eukaryotic initiation factor 2.

The mechanism for guanine nucleotide exchange with eukaryotic initiation factor-2 (eIF-2) from Drosophila melanogaster embryos was studied using the reaction eIF-2 X [3H]GDP + GDP (GTP) in equilibrium eIF-2 X GDP (GTP) + [3H]GDP. When highly purified eIF-2 is used the rate of nucleotide exchange is greatly reduced by Mg2+ and this reduction is overcome by the guanine-nucleotide-exchange factor (GEF) of rabbit reticulocytes. This GEF-dependent exchange is inhibited when Drosophila eIF-2 is either phosphorylated by the hemin-controlled inhibitor (HCI) of rabbit reticulocytes or treated with phosphatidylserine or a rabbit eIF-2 X phosphatidylserine complex. The Mg2+ impairment of guanine nucleotide exchange is less severe when highly purified eIF-2 is incubated at a higher temperature (37 degrees C) and is not observed at any temperature if partially purified eIF-2 is used instead of the highly purified factor. In the latter two cases the exchange is not inhibited by either phosphorylation with HCI or phospholipid treatment of Drosophila eIF-2, possibly suggesting that the observed exchange is not mediated by a GEF-like factor. Our data support two possible mechanisms for GDP/GTP exchange with Drosophila embryos eIF-2: a GEF-dependent exchange, similar to that described in rabbit reticulocytes, which may be regulated by phosphorylation of eIF-2, and a factor-independent exchange which appears to be insensitive to this type of control.

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.

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.