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>.