Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity

Three-dimensional (3D) models of FMDV 3A protein dimer (primary UniProtKB/TrEMBL accession code Q9DJC8) and 3A mutants Q44R, Q44D, M29R, L38E, and L41E were generated using comparative modeling procedures. As a structural template, the soluble domain of human PV 3A protein was used, attending to its hypothetical structural similarity to the target sequence (Protein Data Bank code 1NG7) (65). Best representative conformer in the NMR spectroscopy ensemble was selected as a source of coordinates for the FMDV 3A wild type and subsequent models. All models were built using the SWISS-MODEL server (31, 51, 59), available at http://swissmodel.expasy.org//SWISS-MODEL.html, and their structural quality was checked using the analysis programs provided by the same server (Anolea/Gromos). Global model quality estimation was performed in terms of QMEAN4 raw score (10). To optimize geometries, models were energy minimized using the GROMOS 43B1 force field implemented in DeepView (31), using 500 steps of steepest descent minimization followed by 500 steps of conjugate-gradient minimization. Figures were generated using the Pymol Molecular Graphics System (Schrödinger, LLC). Structurally based sequence alignment of wild-type model templates and other 3A proteins was refined with T-COFFEE (46).

The N-terminal region of 3A, spanning residues I1 to F52, as well as four analogues with the M29R, L38E, L41E, and L38E-L41E replacements (N-ter peptides; see Table 1), were made in C-terminal carboxamide form on Rink amide ChemMatrix resin in a Prelude synthesizer (Protein Technologies, Tucson, AZ). Standard 9-fluorenylmethoxy carbonyl (Fmoc) protocols at a 0.05-mmol scale were used, with 5-fold molar amounts of Fmoc-amino acids and O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 10-fold molar amounts of N,N-diisopropylethylamine, with systematic double coupling for all residues. After deprotection and cleavage with trifluoroacetic acid-water-triisopropylsilane (95:2.5:2.5 [vol/vol], 90 min, room temperature), peptides were precipitated with chilled diethyl ether, redissolved in water, lyophilized, and purified by preparative reverse-phase high-performance liquid chromatography (HPLC) to a minimum purity of 95% by analytical HPLC. Their identities were confirmed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry in a Voyager DE-STR instrument (Applied Biosystems), using α-cyano-4-hydroxycinnamic acid matrix and linear mode acquisition (Table 1).

For SDS-PAGE analysis, aliquots containing 1.87 μg and 0.187 μg of each peptide were prepared from 5-mg/ml stock solutions of the lyophilized peptides in Milli-Q water, mixed with Laemmli sample buffer containing 2% SDS and 100 mM dithiothreitol (DTT), and incubated for 25 min at room temperature. Samples were resolved on 12% SDS-PAGE gels, stained with Coomassie blue, transferred to nitrocellulose membranes, and immunoblotted with 3A-specific rabbit polyclonal antibody (Ab) and a goat anti-rabbit horseradish peroxidase-coupled secondary Ab (GE Healthcare). Samples were subsequently developed using a chemiluminescence kit (Perkin-Elmer).

Vero, IBRS-2, and BHK-21 cells were grown at 37°C and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL), supplemented with 5% fetal bovine serum (Gibco-BRL), 1 μg/ml streptomycin, and 1 μg/ml penicillin. A viral stock from a type C FMDV C-S8c1 (61) isolate was produced by amplification in BHK-21 cells. Procedures for infections and virus titration in semisolid agar medium were as described previously (54).

Monoclonal Ab (MAb) 2C2 to nonstructural protein 3A (21) was used in the proximity ligation assay. Rabbit polyclonal anti-β-tubulin 196 was used in Western blot analysis (4). Anti-rabbit IgGs secondary Ab coupled to Alexa Fluor (AF) 647, used in immunofluorescence assays, was from Molecular Probes (Invitrogen). Polyclonal Ab 443 against the N terminus of 3A protein was produced by immunizing two New Zealand White rabbits with a synthetic peptide spanning 3A residues F26 to Q43 (FEGMVHDSIKEELRPLIQ) coupled to Cys-KLH. To generate polyclonal Ab 346 against the C terminus of 3A, plasmid pRSET-C (Invitrogen) was used for the inducible isopropyl-β-d-thiogalactopyranoside (IPTG) expression in Escherichia coli BL21(DE3)pLysS of a His-tagged polypeptide spanning 3A residues 81 to 153 fused to 3BBB, whose sequence was amplified from the pMT28 clone with primers 5′-TAGGCGCTAGCAGACAGAAGATGGTG-3′ and 5′-GCAGATCTTTACTCAGTGACAATCAA-3′. The restriction enzymes NheI and BglII (New England Biolabs) were used for cloning. The His-tagged protein was purified in an Ni column (Probond resin; Invitrogen) with a pH 4.5 elution buffer, and two New Zealand White rabbits were immunized with the purified protein. Rabbit immunizations were as described previously (54).

To assess their effect on FMDV infectivity, 3A mutations were introduced in plasmid pMT28 encoding the type C FMDV isolate C-S8c1 full-length sequence (29). Substitutions of selected amino acids were performed by site-directed mutagenesis (41), using the primers shown in Table 2. To this end, a 3A-containing fragment was amplified from pMT28 using primer 3A-F and the corresponding mutated reverse primer (-R) or primer 3A-R and the corresponding mutated forward primer (-F) at a 1 μM concentration, in a reaction, including the Expand high-fidelity PCR system BioTaq (Bioline) polymerase (1.25 U), plus Pfu (Biotools) polymerase (0.15U), 2 mM MgCl₂, and 1 mM deoxynucleoside triphosphate (dNTPs) (Roche). After amplification, each PCR fragment was purified and an overlap extension PCR was performed using outer primers 3A-F and 3A-R. The resulting amplicons were digested with BglII and RsrII and cloned into pMT28 digested with the same restriction enzymes, as described previously (40). The correct orientation and sequence of the resulting plasmids were confirmed by sequencing with primers 3A-F and 3A-R (Table 2), as well as primers 3A-1 and 3ABB-2 (30). For transient expression, plasmid pRSV3Awt (30) and derivatives of pRSV/L in which the luciferase gene was replaced by the 3A coding sequence of the different mutants described above were used. To this end, 3A-containing sequences were amplified from the corresponding pMT28 derivatives using oligonucleotides 3A-1 and 3A-2 (30), which included restriction sites for cloning into pRSV (HindIII and KpnI).

For RNA synthesis, plasmids were linearized with NdeI (New England BioLabs) and in vitro transcribed using SP6 RNA polymerase (Promega). After transcription, the reaction mixture was treated with RQ1 DNase (1U/μg RNA; Promega). Then, RNA was extracted with phenol-chloroform and precipitated with ethanol. The RNA integrity and concentration were determined by electrophoresis on agarose gels. In vitro-transcribed RNAs were transfected into BHK-21 cells using the Lipofectin reagent (Invitrogen), as described previously (40). Cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM)-supplemented fetal bovine serum (FBS). To assess RNA infectivity, at 4 h posttransfection (hpt) cells were maintained in semisolid DMEM-0.5% agar supplemented with 5% FBS. At 24 hpt, the viral titer was determined by plaque assay (54).

Viral RNA was extracted from supernatants of cell cultures or tissue homogenates from suckling mice (7), using TRI reagent (Sigma), as described by the manufacturer. cDNA was synthesized by reverse transcription of viral RNA using M-MuLV reverse transcriptase (Roche) and primers 3A-F and 3A-R. cDNA was amplified by PCR using the same primers and BioTaq DNA polymerase (Bioline) supplemented with 10% of Expand high-fidelity polymerase (Biotools) for proofreading activity. PCR products were purified with Wizard SV gel and PCR clean-up system (Promega) and their sequences determined by automatic DNA sequencing. DNA sequences, spanning the nucleotides corresponding to residues 105 of 2B to 5 of 3B2 viral proteins, were confirmed by at least two independent sequencing reactions using the primers indicated above. Nucleotide positions correspond to those previously described for the FMDV C-S8c1 isolate (GenBank no. AJ133357).

Rabbit lysate reticulocytes (Promega) were incubated with 1 μg of transcribed RNAs and 14.3 μCi of [³⁵S]methionine and cysteine (Amersham) for 1 h at 30°C and treated with 50 μg of RNase A for 15 min at room temperature, as described previously (56). Translated proteins were analyzed by 10% SDS-PAGE.

Samples were processed as described by the manufacturer (OLINK Bioscience). Briefly, IBRS cells were grown on glass coverslips and transfected with 1 μg of pRSV3Awt and derivatives using Lipofectamine (Invitrogen) as described by the manufacturer or infected with FMDV C-S8c1 at a multiplicity of infection (MOI) of 5 PFU/cell. At 24 hpt, or 3 h postinfection (hpi), monolayers were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min at room temperature, blocked, and permeabilized with PBTG buffer (0.1% Triton X-100, 1% bovine serum albumin [BSA], and 1 M glycine in PBS) for 15 min at room temperature. Primary Ab was prepared using the Probemaker kit (OLINK, Bioscience) by using the manufacturer's recommendations: 1 mg/ml of monoclonal Ab (IgG class) 2C2, affinity purified through a protein G column, was independently conjugated to each of a pair of oligonucleotides to generate plus and minus proximity ligation assay (PLA) probes. Cells on coverslips were incubated with conjugated primary antibodies diluted in PBS with 1% BSA for 1 h at room temperature and then washed twice with buffer A (0.01 M Tris, 0.15 M NaCl, and 0.05% Tween 20) for 5 min. The signal development (ligation, amplification, and hybridization) was performed according to the manufacturer's instructions in PLA Duolink II. Briefly, a ligation-ligase solution was added to each sample, and slides were incubated in a preheated humidity chamber for 30 min at 37°C and washed twice in buffer A for 2 min with gentle agitation. Then, the amplification-polymerase solution, including the fluorescent probe (λ_exc of 554 nm), was added and slides were incubated in the preheated humidity chamber for 100 min at 37°C. Finally, the slides were washed three times in buffer B (0.2 M Tris, 0.1 M NaCl [pH 7.5]) for 10 min with agitation and protected from light. In the case of transiently transfected cells, samples were further incubated with primary polyclonal Ab 346 and anti-rabbit IgG secondary antibodies coupled to Alexa Fluor 647 (Invitrogen) to detect 3A protein. Slides were mounted with a coverslip using a minimal volume of Duolink II mounting medium with DAPI (4′,6-diamidino-2-phenylindole). Cells were observed with a confocal LSM710 vertical (Bio-Rad/Zeiss) microscope. As reported for dimerization detection by PLA (45), fluorescence was quantified using the ImageJ software (analyze particles plug-in). The percentage of the fluorescence intensity, relative to that determined for cells expressing 3Awt protein, was plotted for each mutant protein ± standard error.

Vero cells were grown on 35-mm tissue culture plates and transfected as described above with 1 μg of different plasmids. At 24 hpt, the cells were scraped on ice into NP-40 lysis buffer (10 mM EGTA, 2.5 mM MgCl₂, 1% NP-40, 20 mM HEPES [pH 7.4]) and sonicated. Equal amounts of total protein of each sample, estimated by Bradford (13), were mixed with Laemmli sample buffer and boiled. Samples were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked, and proteins were detected by incubation with the corresponding 3A-specific polyclonal rabbit Ab as described above.

The infectivity of the FMDV 3A mutants in suckling mice was tested as described previously (7). Briefly, in vitro-transcribed viral RNA (from 10² to 10⁵ ng) mixed with 20 μl of Lipofectin (Invitrogen) in a final volume of 100 μl in PBS was inoculated intraperitoneally in litters (4 to 5 mice per RNA) of Swiss newborn mice. Dead animals were scored up to 11 days after infection. Mice showing severe signs of disease (tremors ataxia, paralysis of the hind limbs) were euthanized. All animals in this study were handled in the BSL-3 facilities at CISA-INIA (Madrid, Spain), in strict accordance with the guidelines of the European Community 86/609/CEE. The protocol was approved by the Committee on the Ethics of Animal Experiments of INIA (permit number CBS 2008/016).

One-way analysis of variance was performed with statistical package SPSS 19.0 (SPSS, Inc.) for Windows. Data are presented as means ± standard errors.

To provide basic insights into the structure-function relationships of the FMDV 3A protein, 3D structural models of its N-terminal region (residues 1 to 94) were generated by bioinformatic procedures, using the analogous structure of the soluble domain of human poliovirus 3A protein (65) as the template. As a result, compatible ungapped models were rendered for residues 15 to 48 of the FMDV 3A protein, in correspondence to the structured region in solution from the experimental template. Quality QMEAN4 raw scores for the model of wild-type protein monomers in this region were 0.622 (Z score = 0.09) and 0.621 (Z score = 0.09), respectively. These values are comprised within the range of quality standards for accepted homology-based structure models (10). The tertiary structure of each monomer is organized mainly on the structural basis of two α-helices (α1, residues 25 to 33, and α2, residues 37 to 44) connected by a 3-residue loop (Fig. 1A and B), hypothetically responsible for the dimerization process. Consensus from diverse methods of transmembrane domain predictions (6, 33, 70) points to the region comprised between residues 59 to 76 as putatively responsible for 3A protein anchorage to membrane (Fig. 1A). General inspection of a minimized 3D structure of the wild-type FMDV 3A homodimer model showed a quaternary organization of the N-terminal region protein similar to that of PV 3A protein obtained by NMR spectroscopy (65). The dimerization interface in FMDV 3A would include all residues in the 25-to-44 stretch of the two antiparallel monomers. As in PV 3A, a number of hydrophobic contacts between α1 and α2 helices could provide physical stability to the dimer. Such interactions would involve residues F25, F26, M29, V30, L38, L41, and I42, in both monomers. Genotypes carrying mutations introducing similar electrostatic repulsive interactions in the hydrophobic core could be unable to be functionally competent. In addition, gradations of that biological effect would be also related to the differential local accumulation of heterogeneous repulsive interactions. Thus, mutations L38E, L41E, and M29R within the hydrophobic core could disrupt the binding interaction in the region by creating repulsions from facing negatively (or positively) charged residues (Fig. 1C). On the other hand, polar contacts such as that of residues D32 and Q44 (Fig. 1C), flanking the C-terminal ends of α1 and α2 helices and exposed to the solvent, could also modulate the interactions between both monomers. Mutation Q44R, found to mediate FMDV adaptation to the guinea pig (48), could significantly modify the electrostatic surface of the region, favoring the formation of salt bridges between both R44 and D32 residues and eventually increasing dimer stability.

Based on these predictions, residues at positions 29, 38, and 41, highly conserved among FMDVs of different serotypes (16), were selected to be replaced by a negatively charged E residue, which would interfere in the hydrophobic interactions predicted in this 3A region. On the other hand, two replacements at residue 44 were analyzed: Q44R and Q44D, predicted to influence dimer stability.

Identification of protein bands potentially corresponding to 3A dimers in FMDV-infected cells is impaired by the presence of protein precursors from the 3ABBB region (30). Therefore, we first addressed the effect of the substitutions selected on FMDV 3A protein transiently expressed in cultured cells. In cells transfected with plasmid pRSV3Awt, a major protein band of the expected mobility was observed by Western blotting using a 3A-specific Ab (Fig. 2A). Additional lower-mobility bands suggestive of 3A dimer/multimer formation were also observed. When cells were transfected with pRSV derivatives expressing 3A with mutations M29R, L38E, or L41E or double-mutant L38EL41E, expected to destabilize dimer formation, no bands corresponding to putative dimers were observed (Fig. 2A). Dimerization was also observed in cells expressing mutant proteins Q44D and Q44R, with the intensity of the dimer band in both cases slightly lower than that of 3Awt.

To further characterize 3A homodimerization, we set up an in situ protein ligation assay previously used to visualize protein-protein interaction in the cell by fluorescence microscopy (3, 28, 45). The strategy, detailed in Materials and Methods and summarized in Fig. 3A, is based on coupling a monoclonal Ab that recognizes a single epitope on each 3A molecule to two different oligonucleotide probes, + and −, which subsequently hybridize to oligonucleotides included in a ligation solution. Should the pair of antibodies locate close enough (<40 nm), their attached probes would ligate, forming a circle; amplification of this circle upon addition of fluorescently labeled nucleotides allows visualization of each single interaction by fluorescence microscopy (71). The monoclonal Ab selected for this assay was directed against an epitope on the C-terminal region of 3A, distant from the N-terminal region analyzed to avoid loss of recognition due to the mutations introduced. The assay included a biological control of noninfected or transfected cells and two technical controls lacking one of the probes each. As shown in Fig. 3B, when cells were FMDV infected and incubated with probes 2C2+ and 2C2−, a dimer fluorescent signal was observed. Fluorescence was also noticed in cells transfected with pRSV3Awt. Significant reductions in the fluorescence intensity, of about 40% of that of 3Awt, were detected in cells transfected with mutant plasmids pRSV3AL38E and pRSV3AL41E (Fig. 3C and D). When plasmid pRSV3AL38E-L41E bearing the two substitutions was analyzed, the signal reduction observed was higher, at about 80% (Fig. 3C). These results support the capacity of 3A to form homodimers and suggest that replacements L38E and L41E impaired dimer formation.

To study the potential of the N terminus of 3A to form dimers and multimers, peptides spanning residues I1 to F52 (termed N peptides) were synthesized by solid-phase procedures, as described in Materials and Methods. The electrophoretic mobility of peptide N-wt, spanning the sequence corresponding to the parental C-S8c1 FMDV, revealed by Western blotting with an Ab against the N terminus of 3A protein, showed a band of a size about that corresponding to the monomeric peptide, as well as additional bands corresponding to higher-order forms (Fig. 2B). A pattern of bands similar to that observed by Western blotting was revealed by mass staining; in this case, the monomeric form was the major band, indicating that the Ab used preferentially recognized dimer/multimeric peptide forms. The inverse relationship observed between the higher-order forms revealed by the Ab recognition or by mass staining could be related to an increase of Ab recognition due to epitope multimerization. On the other hand, only monomeric bands were detected by mass staining of mutant peptides, which showed slight differences in mobility, as reported to occur among 48-mer peptides differing in single residues (53). In addition, the Ab staining showed a dramatic decrease of higher-order forms, N-M29R being the only peptide for which a faint dimeric band was observed. The poor Ab recognition of peptide N-L38E could be explained by either an altered conformation adopted by this peptide or by a possible implication of the mutated residue in the peptide-Ab interaction.

Thus, the overall results indicate that (i) FMDV 3A protein can form dimers and multimers, (ii) its N-terminal fragment is involved in this process, and (iii) replacement of the hydrophobic residue L at positions 38 and 41 by a charged amino acid E diminishes dimerization.

To assess the effect of replacements L38E and L41E located at the predicted hydrophobic dimerization interface on the FMDV life cycle, the infectivity of mutant RNAs transcribed from plasmids pMT28L38E and pMT28L41E carrying these substitutions was compared with that of the corresponding parental C-S8c1 RNA derived from plasmid pMT28. To this end, plaque formation was determined after 24 h of transfection with different amounts of RNA (from 1 to 10³ ng) of BHK-21 cell monolayers maintained in semisolid medium. No infectious virus was recovered after transfection of cells with up to 1 μg of RNA transcribed from pMT28L38E or pMT28L41E (data not shown), indicating that RNAs with these mutations showed an infectivity at least five log lower than that of the RNA control pMT28. When transfected cells were incubated in liquid medium, a delay in the recovery of infectious virus, relative to transcript pMT28, was observed for transcripts pMT28L38E and pMT28L41E (no infective virus was found at 24 hpt, and limited titers of about 1 × 10² and 10 PFU/ml were detected at 72 hpt, respectively) (Fig. 4). Upon two additional passages of the transfection supernatant, the viral titers recovered from transcripts pMT28L38E and pMT28 were similar, while those recovered from transcript pMT28L41E were about 1 log lower (Fig. 4). Emergence of infectious virus was concomitant with the imposition in the viral populations of replacements for each of the mutant RNAs analyzed—E38V and E41A—that restored the hydrophobicity of the residues, as determined by sequencing of the viral RNA at the second cell passage given to the transfection medium (Table 3). Selection of mutant E38V was observed in the two additional transfections of RNA from pMT28L38E performed. On the other hand, infectious virus was recovered in only one of the three additional transfections carried out with RNA from pMT28L41E, in which replacement E41A was found imposed on the viral populations (Table 3). These results indicate that replacement of hydrophobic residues by charged residues at positions 38 and 41 of 3A drastically reduces viral multiplication, leading to selection of substitutions to nonpolar V and A residues that restore hydrophobicity and favor dimer stability.

To study whether the point mutations introduced in 3A could alter the translation of the viral polyprotein, in vitro-transcribed RNAs from pMT2-L38E and pMT28L41E were in vitro translated in a rabbit reticulocytes lysate. The protein pattern obtained for the different mutants was similar to that of pMT28 control RNA (data not shown).

To study the effect of electrostatic charge acquisition at residue Q44 on FMDV life cycle, the infectivity of mutants with replacements Q44R and Q44D, which resulted in a slightly lower dimer detection in transiently expressed 3A mutants (Fig. 2A), was analyzed. An infectivity of about 197 PFU/ng of RNA was determined for transcript pMTQ44R, a value slightly higher than that observed for the control RNA from pMT28 (data not shown). When transfected cells were incubated in liquid medium, RNA from mutant Q44R produced a cytopathic effect at a time similar to that of pMT28, comparable viral titers were found in the culture medium at 24 hpt and upon the two additional passages of the transfection supernatant (Fig. 4), and the viruses recovered maintained the substitution over 3 additional cell passages. Similar results were obtained from two additional transfections performed with pMTQ44R RNA. These results confirm that replacement Q44R does not exert a detrimental effect on FMDV infectivity in cultured cells.

On the contrary, an infectivity of <1 PFU/μg was found for RNA from pMTQ44D, a delay in the recovery of infectious virus was observed after its transfection (no infective virus was found at 24 hpt, and limited titers of about <10 PFU/ml were detected at 72 hpt), and the viral titers recovered after two passages of the supernatant of cells transfected were about 1 log lower than those recovered from pMT28 RNA. Emergence of infectious virus was associated with the imposition of replacement D44E, which was also observed in three additional transfections of RNA from pMT28-Q44D (Table 3).

On the other hand, in vitro translation of RNAs from pMT28Q44R and pMT28Q44D showed a protein pattern similar to that of pMT28 control RNA (data not shown). Thus, while replacement Q44R leads to viruses with infectivity similar to that of the parental C-S8c1 virus, introduction of an acidic residue at position 44 (replacement Q44D) imposes selection of an E residue that maintains the negative charge but acquires a structure similar to that of the parental Q.

To assess the effect of the mutations introduced in 3A on the replication of the virus in an animal model, RNAs transcribed from pMT28L38E, pMT28L41E, pMT28Q44R, and pMT28Q44D were injected into suckling mice, and the emergence of clinical signs and instances of death were followed for 10 days. RNA from pMT28 was used as a control. An amount of 100 ng RNA from pMT28 killed 4 of the 5 animals injected, a lethality value similar to that reported for other FMDV RNAs derived from full-length infectious clones (7). RNA from pMT28-Q44R was also lethal for suckling mice, as 100 ng of RNA killed 3 out of 4 injected animals, and sequencing of the 3A coding region of RNA extracted from dead mice showed the presence of replacement Q44R, confirming the capacity of the virus carrying this mutation to infect suckling mice (47). Conversely, RNAs from pMT28Q44D, pMT28L38E, and pMT28L41E were not lethal at doses of RNA up to 10⁵ ng (Table 4), suggesting that these replacements allow viral replication in cultured cells, enabling selection of revertant mutants, and abolish viral multiplication in an animal model, such as suckling mice.