Selection of functional 2A sequences within foot-and-mouth disease virus; requirements for the NPGP motif with a distinct codon bias

Foot-and-mouth disease virus (FMDV) has a positive-sense ssRNA genome including a single, large, open reading frame. Splitting of the encoded polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues long), which induces a nonproteolytic, cotranslational cleavage at its own C terminus. A conserved feature among variants of 2A is the C-terminal motif N(16)P(17)G(18)/P-19, where P-19 is the first residue of 2B. It has been shown previously that certain amino acid substitutions can be tolerated at residues E-14, S-15, and N-16 within the 2A sequence of infectious FMDVs, but no variants at residues P-17, G(18), or P-19 have been identified. In this study, using highly degenerate primers, we analyzed if any other residues can be present at each position of the NPG/P motif within infectious FMDV. No alternative forms of this motif were found to be encoded by rescued FMDVs after two, three, or four passages. However, surprisingly, a clear codon preference for the wt nucleotide sequence encoding the NPGP motif within these viruses was observed. Indeed, the codons selected to code for P-17 and P-19 within this motif were distinct; thus the synonymous codons are not equivalent.