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.