Picornavirus evolution: genomes encoding multiple 2A^NPGP sequences—biomedical and biotechnological utility

Alignment of picornavirus proteinase/polymerase sequences reveals this family evolved into five ‘supergroups’. Interestingly, the nature of the 2A region of the picornavirus polyprotein is highly correlated with this phylogeny. Viruses within supergroup 4, the Paavivirinae, have complex 2A regions with many viruses encoding multiple 2A^NPGP sequences. In vitro transcription/translation analyses of a synthetic polyprotein comprising green fluorescent protein (GFP) linked to β-glucuronidase (GUS) via individual 2A^NPGPs showed two main phenotypes: highly active 2ANPGP sequences—similar to foot-and-mouth disease virus 2ANPGP—and, surprisingly, a novel phenotype of some 2A^NPGP sequences which apparently terminate translation at the C-terminus of 2A^NPGP without detectable re-initiation of downstream sequences (GUS). Probing databases with the short sequences between 2A^NPGPs did not reveal any potential ‘accessory’ functions. The novel, highly active, 2A-like sequences we identified substantially expand the toolbox for biomedical/biotechnological co-expression applications.