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J. Biol. Chem., Vol. 278, Issue 42, 41019-41027, October 17, 2003

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Complete Alanine Scanning of Intersubunit Interfaces in a Foot-and-Mouth Disease Virus Capsid Reveals Critical Contributions of Many Side Chains to Particle Stability and Viral Function^*

Roberto Mateo , Ana Díaz, Eric Baranowski  and Mauricio G. Mateu ¶

From the Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received for publication, May 13, 2003 , and in revised form, June 25, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Spherical virus capsids are large, multimeric protein shells whose assembly and stability depend on the establishment of multiple non-covalent interactions between many polypeptide subunits. In a foot-and-mouth disease virus capsid, 42 amino acid side chains per protomer are involved in noncovalent interactions between pentameric subunits that function as assembly/disassembly intermediates. We have individually truncated to alanine these 42 side chains and assessed their relevance for completion of the virus life cycle and capsid stability. Most mutations provoked a drastic reduction in virus yields. Nearly all of these critical mutations led to virions whose thermal inactivation rates differed from that of the parent virus, and many affected also early steps in the viral cycle. Rapid selection of genotypic revertants or variants with forward or compensatory mutations that restored viability was occasionally detected. The results with this model virus indicate the following. (i) Most of the residues at the interfaces between capsid subunits are critically important for viral function, in part but not exclusively because of their involvement in intersubunit recognition. Each hydrogen bond and salt bridge buried at the subunit interfaces may be important for capsid stability. (ii) New mutations able to restore viability may arise frequently at the subunit interfaces during virus evolution. (iii) A few interfacial side chains are functionally tolerant to truncation and may provide adequate mutation sites for the engineering of a thermostable capsid, potentially useful as an improved vaccine.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Assembly and stability of the multimeric proteins that constitute icosahedral virus capsids depend on the occurrence of multiple non-covalent interactions between many polypeptide subunits (1, 2). Compared with small oligomeric proteins, the adaptive structural solutions for subunit recognition and stability may be uniquely limited in virus capsids. For example, the many repetitive intersubunit interactions must be strong enough to provide stability against dissociation by extracellular agents, although not so strong as to impair the mechanism of intracellular uncoating. Viral capsids thus provide good models for understanding protein-protein recognition evolved under stringent, even conflicting, selective constraints (3–7).

Structural studies of many viruses (see Refs. 1–5 and 8–10 for some reviews and Ref. 11 for the VIPER data base of capsid structures) and structure-function analyses involving mutation of specific capsid residues (see Refs. 12–24 for some representative studies) have greatly contributed to our current understanding of the assembly, disassembly, and stability of viruses. For small protein oligomers, comprehensive alanine-scanning mutagenesis analyses (25) on the role of interfacial residues in association (26–30) have been carried out, and a computational analysis of protein-protein interactions in icosahedral viruses has been also described (31). However, to our knowledge no systematic mutagenesis approach to experimentally dissect the contribution of each residue in the subunit interfaces to the stability or functionality of a virus capsid or any other large protein assembly had been attempted to date.

Foot-and-mouth disease virus (FMDV),¹ a picornavirus for which much structural and functional information is available, provides a simple model for the dissection of the molecular determinants of assembly, stability, and disassembly of viral capsids and other large protein complexes. Moreover, FMDV causes the economically most important disease of farm animals (32). The recent epizootics in the United Kingdom, with estimated losses that could exceed £10 billion in that country alone (33), have provided a stark remainder within the European Union of the devastating economic and social damage FMDV may cause worldwide. One important problem of the current FMD vaccines, based on chemically inactivated virions, is their very low stability. Vaccines based on particles with improved thermostability would be highly desirable in the prevention of FMD and other viral diseases.

Assembly of the picornaviral capsid proceeds in several steps (34). The capsid proteins VP0 (1AB), VP3 (1C), and VP1 (1D) are translated as a polyprotein precursor (P1), may fold co-translationally (2), and are proteolytically processed to yield the mature protomer. Five protomers are assembled to form a pentameric intermediate, and finally, 12 pentamers are assembled to form the icosahedral capsid (Fig. 1). After encapsidation of the RNA genome most VP0 molecules are processed to give VP4 (1A, the N terminus of VP0) and VP2 (1B). Disassembly of the FMDV virion in vivo begins with its dissociation into pentamers (35) by acidification in the endosomes (36). Mild heating of FMDV virions also leads to irreversible dissociation into stable pentamers (34), an event that appears as the main cause for the need of a cold chain to preserve FMD vaccines. Analysis of the crystal structure of the FMDV capsid (37–41) indicates that the pentameric intermediate subunits interact mainly through a relatively limited number of electrostatic interactions; a role of His-142 of VP3 in the acid-induced disassembly of FMDV has already been demonstrated (20). In the present study, the functional importance of each amino acid side chain involved in noncovalent interactions between subunits in a virus protein shell has been tested by alanine-scanning mutagenesis of the FMDV capsid. The results reveal severe intolerance to variation due to the involvement of many interfacial residues in capsid stability and in other steps of the virus life cycle, and identify those tolerant residues that could be replaced for the rational engineering of a thermostable capsid.

View larger version (49K): [in this window] [in a new window]   FIG. 1. The capsid of FMDV. A, schematic structure of the icosahedral FMDV capsid. VP1 (labeled 1), VP2 (2), VP3 (3), and VP4 (internal) form a biological protomer. Five biological protomers and the pentamer subunit they form are outlined by thick lines. Twelve pentamers form the capsid. The icosahedral symmetry axes are indicated (5x, pentagon; 3x, triangle; 2x, oval). B, structure of a pentamer in the capsid. One biological protomer is represented as a black ribbon model, and its 42 amino acid residues involved in interactions with neighboring pentamers (Table I) are represented as gray space filling models. The remaining four protomers are represented as gray backbone models.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Site-directed Mutagenesis, Subcloning, and DNA Sequencing—Site-directed mutagenesis of FMDV capsid residues was carried out on plasmid p3242/C-S8c1 by the inverse PCR method using the QuikChange system (Stratagene) and pairs of 25- to 36-mer oligonucleotides. The mutations were checked by dideoxynucleotide-based automated sequencing. No second site mutations were found. The mutagenized 3242-bp NgoMIV segments were subcloned in pO1K/3242 to obtain pO1K/C-S8c1 plasmids including the chosen mutations. As a control, a non-mutated 3242-bp NgoMIV segment derived from the original p3242/C-S8c1 plasmid was subjected to the same steps. The entire subcloned 3242-bp segment of a subset of the pO1K/C-S8c1 mutant plasmids, of non-mutated pO1K/C-S8c1, and of a revertant plasmid (obtained by back-mutating Ala-3146 to the original residue) was sequenced. Again no second site mutations were found.

Transcription of Viral RNA and Electroporation of Eukaryotic Cells—pO1K/C-S8c1 and the mutant plasmids were linearized by digestion with HpaI, purified, and dissolved in RNase-free water. Infectious FMDV RNA was transcribed from the linearized plasmids by using the Riboprobe in vitro transcription system (Promega). The mixture contained 40 mM Hepes, pH 7.7, 6 mM magnesium acetate, 2 mM spermidine, 10 mM dithiothreitol, 1 unit/µl RNasin, 2.5 mM each of ribonucleoside triphosphates, 25 ng/µl linearized plasmid DNA, and 0.5 unit/µl SP6 RNA polymerase and was incubated for 2 h at 37 °C. The RNA concentration was estimated by agarose gel electrophoresis. About 2–4 x 10⁶ BHK-21 cells from 60 to 80% confluent monolayers were resuspended in 0.5 ml of electroporation buffer containing 21 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM glucose, and transferred to a Gene Pulser Cuvette (Bio-Rad) with a 0.4-cm electrode gap. FMDV RNA (3 µg) was added and electroporated using a Bio-Rad Gene Pulser set at 280 V, 250 microfarads, 400 ohm (two pulses). The cells were plated in a P60 Petri dish containing 4 ml of Dulbecco's modified Eagle's medium + 10% fetal calf serum and incubated at 37 °C. At 4 h post-transfection the medium was discarded, and 2 ml of fresh Dulbecco's modified Eagle's medium + 2% fetal calf serum was added. The incubation was continued for up to 90 h, with aliquots removed at specified intervals. The progeny virus suspension was clarified by centrifugation, and aliquots were stored at –70 °C. For nearly every transfection experiment, RNA was used immediately after transcription. Use of the same RNA either freshly prepared or frozen and thawed yielded very similar results. The same amount of RNA was used for every mutant. In each experiment the same amount of parent FMDV RNA, and no RNA, was used as a control.

Immunofluorescence Assays—BHK-21 cells were electroporated with viral RNA as described above and subsequently cultured on coverslips. These were washed, incubated with 4% paraformaldehyde, washed again, and incubated with 10 mM glycine buffer, pH 8.5. The cell membranes were permeabilized by incubation with 0.2% Triton X-100 in phosphate-buffered saline (PBS). The coverslips were washed again, incubated in PBS + 3% bovine serum albumin (BSA), and then with monoclonal antibody (mAb) 5C4 (45) (ascitic fluid diluted 1:500 in PBS + 3% BSA), washed thoroughly, incubated with secondary antibody (Alexa-488 from Molecular Probes, diluted 1:500 in PBS + 3% BSA), washed, mounted, and visualized in a fluorescence microscope.

Titration and Amplification of Viruses and Extraction of Viral RNA—Virus titers were determined in plaque assays. Mutant virions obtained at very low titers were amplified by a single passage in BHK-21 cell monolayers at the highest possible multiplicity of infection. The progeny virus suspension was collected immediately after the cytopathic effect was observed, clarified, aliquoted, and frozen at –70 °C. RNA derived from virions collected from the supernatants of transfected or infected cell cultures was extracted using Trizol (Invitrogen) and precipitated with ethanol. The RNA was reverse-transcribed to DNA and amplified by reverse transcriptase-PCR as described previously (46), and the relevant segments were sequenced.

Radioactive Labeling and Purification of Virions—FMDV virions were metabolically labeled with [³⁵S]methionine (Redivue Pro-mix, Amersham Biosciences) and purified by sedimentation through a sucrose cushion followed by sucrose gradient centrifugation, essentially as described (47). The fractions containing full virions (sedimentation coefficient 140 S) were extensively dialyzed against PBS and clarified by centrifugation. The purified virions were free of contaminants, as judged by overloaded SDS-urea-PAGE and Coomassie Blue staining.

Capsid Dissociation Assays and Measurement of Rate Constants— Aliquots (0.3 ml) of ³⁵S-labeled, purified virus were incubated at a constant temperature for different amounts of time, transferred to ice, loaded in 10–30% sucrose density gradients, and centrifuged at 4 °C in an SW40 rotor (Beckman Instruments) at 18,000 rpm for 18 h. The gradients were fractionated in 0.5-ml aliquots, and the radioactivity was determined using a liquid scintillation counter. In a second type of assay, virus suspensions obtained by transfection of infectious mutant RNA were diluted in Dulbecco's modified Eagle's medium + 2% fetal calf serum to a concentration of about 1000 plaque-forming units/ml. 100-µl aliquots in thin walled PCR tubes were incubated at a given temperature (42 °C unless indicated otherwise) for different amounts of time, and the remaining virus titers were determined in plaque assays. Non-mutated C-S8c1 virus obtained in parallel transfection experiments was used in each heat-inactivation experiment as a positive control. The experimental data were fitted to a first-order exponential decay by using the program Kaleidagraph (Abelbeck Software) and Equation 1,

(Eq. 1)

where C is the virus titer at time t; C₀ is the virus titer at t = 0; and k is the dissociation rate constant.

Structural Analyses of Capsid Subunit Interfaces—A silicon graphics work station and the Protein Data Bank coordinates for FMDVs of serotypes C (38) and O (37, 39) were used. The crystallographic models were analyzed using the programs InsightII (Biosym Technologies) and RasMol (48). Contact and solvent accessibility and modeling of mutations were done with the program Whatif (49) using the coordinates of all possible pairs of contacting subunits with different symmetry within the capsid.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Analysis of Interpentameric Interactions within the FMDV Capsid—In the FMDV capsid, interpentamer interfaces involve essentially VP2-VP3 and VP2-VP2 pairwise contacts (37–41) (Fig. 1). In addition, the N termini of three symmetry-related VP2 from different pentamers form a short -annulus around each 3-fold axis (38). The low electronic density associated with this annulus prevented the identification of the few side chains of this element, if any, involved in interactions. The structure of homologous FMDV isolates of three different serotypes, O₁BFS (37, 41), which has been solved to very high crystallographic resolution (1.9 Å), C-S8c1 (38) and A22 (40), revealed high conformational similarity and many conserved interfacial residues and interactions and allowed meaningful contact analyses in the structure of our model FMDV virus (C-S8c1) (38). Within the cut-off distances imposed (Table I), a total of 61 residues per capsid protomer appear involved in direct interpentamer interactions. The side chains of 42 of those residues are involved, beyond C, in such interactions and constitute the interpentamer interface accessible to mutation (Table I and Fig. 1B). Eighteen of these side chains (only 8 being nonpolar) participate exclusively in van der Waals interactions, with an average of 2.8 contacts per residue. Very few (0–2) of these are carbon-carbon contacts (the only exceptions being residues Tyr-2200, Gln-3071, and Thr-2022 with 11, 5, and 3 carbon-carbon contacts, respectively). The remaining 24 polar side chains per protomer at the interfaces are involved in many interpentamer polar interactions. These include, per protomer subunit, a total of 18 different side chain-main chain or side chain-side chain hydrogen bonds, 2 strict salt bridges, 6 medium range charge-charge interactions, and a short range charge-helix dipole interaction (Table I). Most of these 24 side chains participate also in a variable but generally small numbers of van der Waals contacts.

In summary, apart from a limited number of main chain-main chain interactions, essentially all of the direct interpentamer interactions in the FMDV C-S8c1 capsid could be eliminated by truncation to alanine of 42 side chains per protomer that define the pentamer interfaces and that are involved mainly in polar interactions.

Effects of the Truncation of the Side Chains Involved in Interpentameric Interactions on FMDV Infectivity—Each of the 42 interfacial side chains found involved in interpentameric interactions in FMDV C-S8c1 (Table I) was individually truncated to Ala to analyze first its individual role on the completion of the viral life cycle and the production of infectious particles. Mutation to Ala eliminated the targeted side chain beyond C and disrupted any interaction that could involve that side chain, without introducing new interactions and with the lowest probability of altering the conformation of the polypeptide backbone (25, 50). The effect of the introduced mutations on the infectivity of the viral RNA was tested by electroporation of susceptible cells and analysis of the viral progeny (Table II). The non-mutated viral RNA was highly infectious as expected, with complete cytopathic effect being observed at about 35–40 h post-transfection and an average virus titer of 1.6 x 10⁶ plaque-forming units per ml. This titer was obtained with samples collected at 45 h and remained more or less constant up to at least 90 h post-transfection. As a test for the complete mutagenesis step, one of the mutant genomes obtained (E3146A) was back-mutated to wild type (A3146E). As expected, the cytopathic effect, virus titer, and plaque size reverted to normal (Table II).

The 42 mutants could be classified into four groups (Table II). Group I (38% of the mutants) included viruses that could not be recovered at 45 h post-transfection or, in most cases, at longer incubation times. Group II (17%) included mutants that yielded drastically reduced titers at 45 h (about 0.01–0.1% that obtained with the non-mutated genome). These titers increased to different extents in samples taken at longer times (up to 90 h). Group III (14%) was formed by mutants that yielded highly reduced titers at 45 h (about 0.1% to less than 10% that of the wild type) and did not recover at longer times. The remaining mutants yielded titers about 10–100% that of the parent virus. In addition, in most cases the plaques of the progeny virus obtained were clearly reduced in size compared with that of the non-mutated virus (Fig. 2 and Table II). Thus, a remarkable majority of single mutations at the interpentamer interfaces in the viral capsid had a drastic negative effect on the viral life cycle and virus yield.

View larger version (143K): [in this window] [in a new window]   FIG. 2. Large plaque and small plaque virus mutants. Cultured cells were electroporated with no RNA (A), or RNA from non-mutated virus (B), Q2057A, a representative of large plaque mutants (C), or D3148A, a representative of small plaque mutants (D), and titrated in standard plaque assays (see also Table II).

Fixation of Genetic Reversions and Forward Mutations in Viral Progeny Populations—Although several mutants yielded nearly normal titers, this could be the result of reversion rather than tolerance to a specific mutation. Thus, RNA from viral populations recovered at 45 h after transfection with any of those mutants was sequenced. In 8 of 13 cases the consensus sequence of the population, as revealed by the very clean densitograms, showed the introduced mutation was still dominant, with no detected sequence heterogeneity. However, in the remaining 5 cases other mutations had been fixed in the population (Table II). Such mutations included the following. (i) Three cases of genotypic reversion. In one of these cases (E3146A) the densitograms showed a double band at the mutated nucleotide position that indicated the population still contained a sizable fraction of the original mutant. In the two others (E2213A and T3190A), only the revertant genotype was apparent. (ii) Two cases of forward mutations at the position which had been originally mutated: in H2021A and K2063A mutations to Thr or Val had been fixed, respectively. These five site-directed mutants probably had a strongly reduced fitness and were easily outcompeted by revertants or other variants in the population. This left only 19% of the residues at the interpentamer interfaces that are tolerant to mutation (group IV, Table II). Two group I mutants recovered high titers after a long post-transfection incubation time. Sequencing of the global population revealed in one case (N2114A) a heterogeneous mixture of the original Ala mutant and two different variants with forward mutations (Ser and Thr) at the same position. In the second case (N2202A) the population was a mixture of the original Ala mutant and of a double mutant that preserved the original Ala mutation but that included also a second mutation in residue Lys-2063 (to Ile), also at the interpentamer interface, which may phenotypically compensate for the deleterious effect of the original substitution. Interestingly, at least some of the forward mutations that restored viability (N2114T and H2021T) introduced residues that were found in other FMDV field isolates of the widely divergent serotypes SAT2 and SAT3.

The conservation in FMDV of the residues at the pentamer interfaces was evaluated using an alignment of the capsid protein sequences corresponding to 23 isolates from six of the seven FMDV serotypes (Table II). The degree of conservation of the critical or tolerant interfacial residues was 59 or 37%, respectively, numbers that are clearly above or below the average conservation when all VP2 + VP3 residues were considered (45%). However, several interfacial residues critical for the viability of C-S8c1 were not conserved, and several of the substituted residues may not conserve the corresponding interpentamer interactions (see under "Discussion").

Effects of the Truncation of Side Chains Involved in Interpentameric Interactions on the Thermal Stability of Virions— The kinetic stability of the FMDV C-S8c1 capsid against its irreversible dissociation by heat was first analyzed by sedimentation in sucrose gradients using radioactively labeled virions. Non-heated purified virus yielded a peak with a sedimentation coefficient of about 140 S, corresponding to full virions, and another peak of about 12 S corresponding to free pentamers (due to some dissociation during storage of the virus at 4 °C). Heating to a constant, moderate temperature for increasing amounts of time provoked a sharp decrease of the 140 S peak and the simultaneous increase of the 12 S peak. Disappearance of the 140 S peak could be well fitted to a single first-order exponential, consistent with the simple dissociation of virions into pentamers (Fig. 3A). The dissociation rate constant (k_off)of the purified virion at 45 °C in PBS was 0.10 min^–1.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Thermal inactivation of wild-type and mutant virions. A, thermal inactivation curve of FMDV C-S8c1 at 45 °C, as determined by centrifugation of radiolabeled particles in sucrose gradients. B, thermal inactivation at 42 °C of non-mutated and representative mutant virions originally obtained from electroporated cultured cells, as determined in infectivity assays. Circles, non-mutated virus; squares, T2110A; triangles, K2096A; inverted triangles, D3148A. Linear fittings of the experimental data to single exponentials are indicated by solid lines (see under "Results" and Table III).

View larger version (133K): [in this window] [in a new window]   FIG. 4. In situ immunofluorescence detection of viral capsid assemblies in cultured cells. BHK cells were electroporated with RNA from non-mutated virus, with no RNA, or with RNA from the mutants in Table III, as indicated, and formation of capsids or subassemblies was determined using a monoclonal antibody against a discontinuous epitope present in the FMDV capsid. For an interpretation of the results obtained, see Table III and under "Results."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The results obtained demonstrate that in this model virus capsid over 80% of the residues involved in intersubunit (pentamer) interactions are critically important for completion of the virus life cycle. These drastic phenotypic effects appear at least partly related to altered capsid assembly and/or stability against dissociation. The results showed that nearly any mutation tested among those which significantly reduced infectivity, affected also the stability of the virion. In addition to this direct evidence, several other observations are consistent with capsid assembly and/or stability being affected by most of the interfacial mutations. The interfacial residues belonging to any of groups I–III do not cluster at any specific location, as could be expected if these mutations affected any localized function, but are scattered all over the large interpentamer interfaces (Fig. 5). Also, if disruption of an interpentamer interaction is involved in the observed phenotypic effect, it could be expected that mutation of any of the two interacting residues will have similarly critical effects. Such prediction was generally found correct.

View larger version (67K): [in this window] [in a new window]   FIG. 5. Residues involved in interpentamer interactions in the viral capsid and their effect on viability. A, frontal view; B, lateral view of a ribbon model of a protomeric subunit in the FMDV C-S8c1 capsid (38). VP1, VP2, and VP3 are respectively colored cyan, yellow, or magenta. The capsid residues involved in interpentamer interactions are represented as space filling models and color-coded according to their effect in the virus yield. Group I (the most critical residues) is colored red, group II orange, group III yellow, and group IV (tolerant residues) green. Residues for which rapid genotypic reversion or forward mutations occurred are colored pink. C, wire frame model of a pentameric subunit in the FMDV C-S8c1 capsid inclined about 50° from the horizontal and viewed from below (i.e. from the virion interior). Residues tolerant to truncation (group IV) and intolerant residues (all except group IV) are represented as space filling models and respectively colored green or red.

Surface accessibility analysis showed that only three (about one-third) of the non-critical side chains were buried. Non-critical residues were not involved in salt bridges and contributed to just one hydrogen bond (Thr-2053 to Gln-2057), which is fully exposed to solvent on the capsid internal surface, and only one of the tolerant side chains participated in more than two interpentamer van der Waals contacts. In contrast, close to three-fourths of the functionally important interfacial side chains were essentially buried upon assembly. 17 of them (52%) were involved in interpentamer salt bridges and/or hydrogen bonds, and 8 others (24%) participated in multiple van der Waals contacts and/or could be involved in medium range charge-charge interactions (compare Tables I and II). Truncation of any residue involved in interpentamer salt bridges had the most critical effects and led in several cases to genotypic reversion. Truncation of any residue involved in buried interpentamer hydrogen bonds led to a thousand-fold or higher reduction in titer. The above observations are consistent with capsid assembly and stability being dependent in part on the hydrophobic effect and multiple van der Waals contacts between subunits, but mainly on each hydrogen bond and salt bridge buried at the pentamer interfaces.

The individual contribution of interfacial residues to inter-subunit association energies have been theoretically predicted for some virus capsids and the results are available at the VIPER website (11). Our computational approach identified all contact residues, defined as those for which at least one atom is within the cut-off distance from an atom in a neighboring pentamer. Of these residues, only those whose side chain atoms beyond C are involved in contacts (Table I) are, by definition, amenable to mutational analysis by alanine scanning to determine the specific contributions of their side chains. On the other hand, the contact residues included in the VIPER analyses were those whose C is within cut-off distance from the C of a residue in a neighboring subunit. Also, the VIPER prediction of association energies considered the complete residue, not just the side chain, and for FMDV it was carried out with a strain of a different serotype (O₁BFS). Despite these differences, most residues at the interpentamer interfaces were conserved in both strains and were included both in VIPER's analysis and in our analysis. For these interfacial residues, a comparison of the predictions of the VIPER site with our experimental phenotypic study has revealed that mutation of the residues predicted as energetically important (association energy between –1.3 and –3.5 kcal/mol) led in most cases (61%) to drastic phenotypic alterations, whereas only 28% of them led to minor or no phenotypic effects (groups III and IV). In contrast, mutation of the residues predicted as energetically less important (association energy between 0 and –1.0 kcal/mol) led to the most drastic phenotypic alterations in a substantially lower, although still remarkable, number of cases (38%), whereas the proportion of mutations with minor or no effects on the phenotype was raised to 38%.

It may seem surprising that such a large proportion of interfacial residues, many of them involved in interactions that could be considered energetically important for protein-protein association but some of them less so, may individually contribute to capsid assembly and stability. One possibility regards the added effects of the many identical interactions present in a virus capsid (in the case of FMDV, each interaction has a multiplicity of 60), and cooperativity effects may be also important. A theoretical equilibrium thermodynamic model by Zlotnick (52) predicted that loss of even one contact with a small binding energy may cause a significant change in the stability of an icosahedral virus capsid, because of the large number of protomers involved. Our experimental results are consistent with this prediction. A second, non-exclusive possibility regards the fact that a number of the interfacial residues tested (or their coding nucleotides) are also involved in steps of the life cycle prior to the assembly of protomers and pentamers. The precise steps affected have not been identified yet. A defect in viral entry or productive uncoating is unlikely, because the cells were originally transfected and, furthermore, the mutated residues were located away from the natural receptor-binding site. All of the capsid coding region could be deleted without any apparent effect on the ability of the RNA to replicate (53). No involvement of capsid sequences in the control of translation of FMDV proteins has been found either. The position of the mutated residues and the putative structure of the unprocessed picornaviral protomer (2) make their participation in polyprotein processing or RNA-protein interactions also unlikely. Few of the side chains analyzed were involved in substantial intraprotein, intraprotomer, or intrapentamer interactions. Thus, putative defects in protein folding and protomer or pentamer assembly would appear difficult to invoke as a general explanation for the results obtained. Further experiments are required for the identification of these additional defects, which could be different for each mutation. Taken together, the results indicate that both an effect on capsid assembly and/or stability and, in many cases, further effect(s) on steps of the viral life cycle previous to pentamer assembly account for the functional importance of the vast majority of side chains involved in intersubunit contacts.

The observed intolerance to individual truncation of most interfacial side chains in the FMDV capsid is consistent with a degree of conservation of critical residues in natural isolates that was found higher than average. However, a number of the residues identified as critical were not conserved in FMDV. Replication of the mutant viral RNAs in cultured cells led not only to simple reversion events but also to the fixation of new amino acid substitutions that restored the infectious phenotype, and that in some cases had been found in distant field isolates of FMDV. Although further study is required, these observations point to the frequent selection during virus evolution of compensatory mutations at the pentamer interfaces. These would provide the virus with an adaptive solution to preserve assembly-competent protein subunits and stable capsids, even when simple reversion would be difficult because of the accumulation of two or more point mutations (see under "Results"). It may also allow the satisfaction of severe structure-function constraints (3, 6) without compromising the adaptive rapid evolution of RNA viruses (54).

From a biotechnological point of view, the side chains at the subunit interfaces of the FMDV capsid that could be considered functionally less relevant, and thus more adequate for introducing potentially stabilizing interactions through mutation, have been now identified (Table II and Fig. 5). In addition, three alanine mutants were found already more stable than the natural virus. The results provide hope that directed evolution or the rational engineering of just one additional buried salt bridge or hydrogen bond (per protomer) through mutation of some of the few tolerant positions identified may also increase capsid stability. An FMD vaccine based on a chemically inactivated virus with such engineered mutations may relax the requirements for a cold chain, without a need to change the well established methods of FMD vaccine production and testing. The capacity of eliciting a broad immune response by the complete viral particles would be preserved, as the several antigenic sites identified in FMDV do not overlap with interpentamer interfaces (6). The use of empty capsids has been considered in the development of new vaccines to avoid the risk of handling infectious virions. Unfortunately, empty capsids of FMDV and other viruses may be less stable than full virions (55), perhaps because of missing nucleic acid-capsid interactions. The engineering of a capsid with improved stability may allow for the compensation for such effects.

	FOOTNOTES

 
^* This work was supported in part by MCyT Grant BIO2000-0408, Comunidad de Madrid Grants 07B/0008/1999 and 08.2/0008.2/2000 (to M. G. M.), and by an institutional grant from Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a predoctoral fellowship from Gobierno Vasco.

Present address: Institut National de la Recherche Agronomique, UMR 1225, Ecole Nationale Vétérinaire, 23 Chemin des Capelles, 31076 Toulouse Cedex 3, France.

^¶ To whom correspondence should be addressed. Tel.: 34-91-3978462; Fax: 34-91-3974799; E-mail: mgarcia{at}cbm.uam.es.

¹ The abbreviations used are: FMDV, foot-and-mouth disease virus; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BHK, baby hamster kidney; FMD, foot-and-mouth disease.

	ACKNOWLEDGMENTS

 
M. G. M. thanks Prof. E. Domingo for previous support and collaboration, for supplying the FMDV infectious clone, and for critical reading of the manuscript. We also thank Dr. E. Brocchi for supplying mAb 5C4 and Drs. C. Escarmís, C. M. Ruiz-Jarabo, N. Verdaguer, L. Menéndez-Arias, and M. Dávila for technical advice.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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