The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ' primary ' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ' cleavage ' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ' cleavage ' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA Gly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ' skip ' from one codon to the next without the formation of a peptide bond.

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In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus (TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high