Effect of replacement R55W in 2C in the release of FMDV RNA from cells.

<p>10⁶ BHK–21 cells were electroporated with 8 µg of either V19–4, V3, V15–9 or WT RNAs. Then the cells were transferred to M24–wells. <b>A</b>, Total viral RNA (intracellular and extracellular) and extracellular RNA were quantified by real–time RT–PCR, at the indicated hours post-electroporation (hpe). The amount of FMDV RNA was normalised to the number of cells seeded in the corresponding wells. The viral RNA (total and extracellular) measured just after electroporation was subtracted from each corresponding value. Note that RNAs encoding R55W in 2C are significantly more efficient in promoting release of viral RNA from cells at 6 hours post–transfection than those expressing wild type 2C (p<0.05; paired <i>t</i>–Test). <b>B</b>, Ratio of FMDV RNA released from the cells (ratio of extracellular viral RNA to total RNA) at 6 hours post–electroporation, determined from the data shown in A. Assays in A and B were carried out in triplicate and standard deviations are given. <b>C</b>, Detection of capsid proteins VP3 and VP1 in mutant FMDVs expressing 2C with or without replacement R55W. 10⁶ BHK–21 cells were electroporated with 8 µg of viral RNA. At 3 hours post–electroporation, supernatants were removed and DMEM without Met and Cys, but supplemented with [³⁵S] Met–Cys, was added. At 4 hours post–electroporation, supernatants were removed and cells were collected to analyse the synthesis of the viral capsid proteins VP3 and VP1. <b>D</b>, Extracellular samples of cell cultures electroporated with the indicated mutant FMDV RNAs, labelled as described in C, were collected at 2 (0-2 hours) and 20 (2-24 hours) hours post–labelling (6 and 24 hours post–electroporation). Samples were analysed by SDS–PAGE (15% acrylamide). VP3 and VP1 were detected at early time (4 to 6 hours post–transfection) in the supernatant of cells transfected with 8 µg of V15–9 or V3 encoding 2C with R55W, but not with the same amount of RNA from constructs encoding wild type 2C. At later times (6 to 24 hours post–transfection), VP3 and VP1 were detected in the supernatant of cells transfected with any of the constructs tested. Procedures are further detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Effect of replacement R55W in 2C on FMDV cytopathology.

<p><b>A</b>, BHK-21 cell-killing assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Herrera1" target="_blank">[40]</a> carried out with either FMDV WT (expressing three VPgs) with the standard 2C (abbreviated as WT) or FMDV WT with substitution R55W in 2C (abbreviated as WT R55W). Serial dilutions of either FMDV WT or FMDV R55W were applied to wells containing 10⁴ BHK-21 cells each. The plot depicts the minimum MOI (PFU/cell) required to cause complete cytopathology (detachment of all cells from the monolayer) at the indicated hours post-infection, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Herrera1" target="_blank">[40]</a>. Results are the average of four independent determinations. Note that the presence of R55W in 2C decreases the MOI needed to cause complete cytopathology for a given time. <b>B</b>, Quantification of cytopathology (percentage of cells detached from the monolayer) in cells with RNA from wild type (WT) or mutants V19-4, V15-9 or V3 expressing either wild type 2C or 2C with substitution R55W. At 8 h post-electroporation the total number of cells and the number of detached cells were counted; a correction was introduced to account for the number of cells that were detached due to the electroporation by parallel measurements with mock-electroporated cells (around 50% of cell detachment). Results are the average of three determinations.</p

Schematic representation of the FMDV genome and of the constructions with one copy of VPg.

<p><b>A</b>, FMDV genome (VPg is the protein covalently linked to the 5′–end of the RNA; PolyC is the internal polycytidylate tract; IRES is the internal ribosome entry site; A(n) is the PolyA at the 3′–end). The genomic region encoding the viral polyprotein is boxed. The viral polyprotein is processed into the different mature proteins indicated in each corresponding box (based in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Ryan1" target="_blank">[15]</a>). Gene 3B (highlighted) encodes 3 different but related copies of FMDV protein–primer VPg. <b>B</b>, Amino acid sequence of VPg₁, VPg₂ and VPg₃ of FMDV C–S8c1. Infectious FMDV clones were constructed either to express VPg₁ (V1), VPg₃ (V3), a chimeric VPg consisting of the first 19 residues from VPg₁ (N-terminus) and the last 4 residues from VPg₃ (C-terminus) (V19–4), or a chimeric VPg containing the first 15 residues from VPg₁ and the last 9 residues from VPg₃ (V15–9). The starting pMT28 plasmid and procedures for the construction are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Recovery of infectivity of FMDV mutants expressing one VPg, upon serial passages in BHK–21 cells.

<p>2×10⁶ BHK-21 cells were transfected with 100 ng (series <i>A</i>) (panels A, B, C) or 500 ng (series <i>B</i>) (panels D, E, F) of the FMDV transcripts indicated in the boxes. Cells and cell culture supernatants were collected at 72 h post-transfection (passage 0). Successive passages were carried out by infecting BHK–21 cells with the viruses from the cell culture supernatant of the previous passage, and samples were collected when cytopathology was complete (generally 20 to 30 hours pi). <b>A</b>, <b>D</b>, Viral infectivity as a function of passage number; plaque development was for 48 hours. <b>B</b>, <b>E</b>, Number of genomic RNA molecules in the cell culture supernatants. <b>C</b>, <b>F</b>, Specific infectivity calculated as the number of PFU per 10¹⁰ viral RNA genomes, from the data given in A, B and D, E. Measurements were carried out in triplicate and standard deviations are given. Procedures for titration of infectivity and determination of viral RNA levels are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Viral proteins expressed by mutant FMDVs encoding either wild type 2C or 2C with R55W.

<p><b>A</b>, Electrophoretic analysis of proteins labelled with [³⁵S] Met–Cys. 10⁶ BHK–21 cells were electroporated with 2 or 8 µg of the indicated viral RNAs; At 3 hours post–electroporation, supernatants were removed and DMEM without Met and Cys but supplemented with [³⁵S] Met–Cys was added. One hour later, cell culture supernatants were removed and cells were collected in loading buffer, proteins were resolved in SDS–PAGE (15% acrylamide), and the gel was dried and exposed. Viral proteins, identified by their reactivity with specific MAbs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales2" target="_blank">[62]</a>, are indicated with an asterisk. <b>B</b>, Close-up view of the bands in A corresponding to 3CD and 3BCD precursors. Wild type FMDVs expressing 3 copies of VPg show three precursors of lower mobility than 3CD that may correspond to 3BCD, 3BBCD and 3BBBCD, respectively. Mutant FMDVs expressing one VPg show only one precursor above 3CD that may correspond to 3BCD. <b>C</b>, Western blot analysis of samples described in A. Proteins were visualised with a specific polyclonal antibody against FMDV 3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales2" target="_blank">[62]</a>. Molecular size markers were run in parallel and their position is indicated on the left. The positions of viral proteins P3, 3CD, and 3D are indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales2" target="_blank">[62]</a>. Note that precursor P3 (3ABCD) from the mutant transcripts expressing one VPg displayed a higher mobility than WT P3. A band above 3CD was detected in some lanes (indicated with an asterisk) that may correspond to precursor 3BCD in V19–4 R55W and to precursors 3BCD, 3BBCD and/or 3BBBCD in FMDV WT, with or without R55W in 2C. <b>D</b>, Proportion of viral polymerase 3D (as percentage of the value obtained in the electroporation with 8 µg of FMDV WT transcript), measured by densitometry of electropherograms as that shown in A. Values are the average of triplicate protein analyses of independent labelling experiments, and standard deviations are given. WT (0.25x) indicates transfections with 2 µg of WT. The assignment of viral proteins was based on reactivity with specific monoclonal antibodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales2" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Perales3" target="_blank">[66]</a>. Procedures are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Relative amounts of mutant FMDV RNA released from cells ^a.

a<p>10⁶ BHK–21 cells were electroporated with 8 µg of FMDV RNA, and the amount of extracellular FMDV RNA at 6 hours post–electroporation was quantitated as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p>b<p>Mutant FMDVs analysed. Their construction is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>, and depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone-0010735-g001" target="_blank">Figure 1</a>; R55W indicates the presence of replacement R55W in 2C.</p>c<p>Viral RNA released per cell for the indicated transcripts. Values are expressed as the percentage of the amount of RNA released per cell in the transfections with FMDV WT. Each value is the average of at least three determinations; standard deviations are given. Relative viral RNA released per cell in mutant FMDVs defective in plaque–forming capacity is below 20% of FMDV WT (calculated from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone-0010735-g005" target="_blank">Figure 5</a>).</p>d<p>Calculated number of infectious particles released per cell for wild type and mutant FMDVs. An average number of 34 PFU are released per cell in infections with FMDV WT (data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone-0010735-g003" target="_blank">Figure 3</a>). The calculation assumes that one FMDV WT infectious particle is equivalent to one PFU, and that the number of infectious particles released by each mutant virus correlates with the viral RNA released.</p>e<p>Plaque–forming capacity of mutant FMDV transcripts (data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone-0010735-g004" target="_blank">Figure 4</a>). Yes indicates the ability of mutant transcripts to develop plaques in 48 h. No indicates that no plaques were observed at 72 hours post–electroporation. Plaque development with V19–4 R55W was only observed at 72 hours post–electroporation. Procedures for plaque assays of viral transcripts are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Restoration of the plaque–forming phenotype in FMDV mutants by replacement R55W in 2C.

<p>RNA transcripts (10 ng for 48 hours of plaque development and 1 ng for 72 hours plaque development) were transfected into 2×10⁶ BHK–21 cells. At 2 hours post–transfection cell culture supernatants were removed and the cells were overlaid with semisolid agar medium. <b>A</b>, Plaque assay of directly transfected FMDV RNA transcripts encoding either wild type 2C (left) or 2C with substitution R55W (right). For mutants V15–9, V19–4 and V3, plaques were detected when 2C included substitution R55W. WT(0.2x) represents transfections with a fifth of the amount used for the rest of the assays. Plaques for mutant V19–4 (R55W) were not detected at 48 hours post–transfection. V3 formed some plaques at 72 hours post–transfection but not at 48 hours post–transfection. The number of plaques formed by V3 was 18–fold lower than the number formed by V3 encoding R55W in 2C. <b>B</b>, Specific infectivity measured as the ratio of the number of PFU at 72 hours post–transfection to the amount of viral transcript used in the electroporation. Assays were carried out in triplicate, and standard deviations are given. Procedures for plaque assays of transfected cells are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p

Mutations in populations V19–4A and V19–4AB after 27 passages in BHK–21 cells ^a.

a<p>The consensus sequence of the entire viral genome was determined for FMDV populations V19–4A and V19–4B after 27 passages in cell culture.</p>b<p>FMDV genomic region analysed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Ryan1" target="_blank">[15]</a>.</p>c<p>Mutations and deduced amino acid substitutions are relative to the sequence of the parental clone FMDV WT (pMT28, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>); residue numbering is according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone.0010735-Baranowski1" target="_blank">[49]</a>. Amino acid residues (single–letter code) are numbered individually for each protein from the N– to the C–terminus. Two residues separated by a bar indicate a mixture of two nucleotides in the population, according to the sequence chromatogram pattern. Procedures for nucleotide sequencing and identification of FMDV genomic regions are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p>d<p>Populations in which the substitutions indicated in ^c were found.</p>e<p>Mutation C4507U (that corresponds to amino acid substitution R55W in 2C) was dominant in V19–4B at passage 2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#pone-0010735-t001" target="_blank">Table 1</a>) and had reverted by passage 27 (highlighted in italics).</p>f<p>Substitution E8K (G5779A) was found repeated in population V19–4 at passage 27 in series A and B (underlined).</p

Possible multiple origins for hybrid strains based on Supernetworks, Polymorphic sites (Figure S1), Parsimony (Figure S2) and Neighbor-Joining (Figure S3) gene trees, PCR-RFLP data [<b>16</b>], [<b>17</b>], <i>COX2</i> sequence data [<b>17</b>] and maximum parsimony analysis of chromosome rearrangements [<b>32</b>].

<p>Five out of six hybridization events are depicted in this figure, AMH and tripe hybrid origins have not been depicted due because they involved secondary hybridization events, in the case of AMH with another <i>S. cerevisiae</i> strain. The putative genetic backgrounds of the parental strains are indicated by squares on the left of each network. Symbols: triangles correspond to chromosome loss; squares to mitochondrial <i>COX2</i> haplotypes; diamonds to chromosome recombination events; rectangles to mutations generating new allele variants; circles to chromosome non-disjunctions. Those depicted in white are referring to events occurring in the <i>S. cerevisiae</i> subgenome of hybrids; in black, in the <i>S. kudriavzevii</i> subgenome; and in grey, those events involving both subgenomes (recombination events).</p

Effect of replacement E8K in VPg on the specific infectivity of V19–4 (R55W).

<p><b>A</b>, Plaque assay with cells transfected with RNA transcripts of WT or the indicated mutant constructs. At 2 hours post–transfection, the cell culture supernatants were removed and the cells were covered with semisolid agar. Plaques were visualized either at 48 or 72 hours post–transfection. Note that substitutions R55W in 2C and E8K in VPg enhanced the plaque-forming capacity of V19–4. <b>B</b>, Specific infectivity (calculated as the ratio of the number of PFUs obtained to the ng of RNA used in the electroporation) for WT and the indicated V19–4 mutants. <b>C</b>, Total and extracellular FMDV RNAs measured at 6 hours after electroporation of BHK–21 cells with WT and the indicated V19–4 mutant RNAs. <b>D</b>, Amount of viral polymerase (3D) determined by metabolic labelling after electroporation of BHK–21 cells with the indicated RNAs, and densitometry of the relevant ptotein band. The results are expressed as percentage of the amount measured following electroporation with WT RNA, which is taken as 100%. Values in B, C, D are the average of 3 determinations; standard deviations are given. Procedures are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010735#s4" target="_blank">Materials and Methods</a>.</p