Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

Abstract

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.

Picornavirus IRES elements: RNA structure and host protein interactions

“…Insertion of the aphthovirus 2A oligopeptide into a polyprotein was sufficient to detect the individual polypeptides, although the upstream 2A product was in molar excess relative to the downstream product http://dx.doi.org/10.1016/j.virusres.2015.01.012 0168-1702/© 2015 Elsevier B.V. All rights reserved. (Donnelly et al, 2001). These results lead to the proposal that 2A was a translational recoding element (CHYSEL for cis-acting hydrolytic element, also referred to as stop/go translation).…”

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

“…Processing of the IntF2A‐based polyprotein is initiated during protein translation, in which peptide bond formation between the last two amino acids of the F2A peptide, that is glycine and proline, is disrupted by the unique translational recoding activity of the F2A sequence (Donnelly et al ., 2001b). It has been hypothesized that the translational recoding event requires specific interaction of the ribosome exit tunnel with the nascent 2A peptide to constrain the conformational space of the peptidyl(2A)‐tRNA gly ester bond in the ribosome P‐site to ‘jam’ further elongation (Minskaia et al ., 2013; Roulston et al ., 2016; Yan et al ., 2010).…”

“…Polyprotein expression based on the unique ribosome skipping mechanism of the FMDV 2A (F2A) peptide (Donnelly et al ., 2001b) operates cotranslationally. The F2A peptide has been used to direct multiprotein co‐expression in a wide range of eukaryotic hosts (de Felipe, 2004; de Felipe et al ., 2006).…”

Self Replicating Gene Vaccine Carrying P1-2A Gene of FMDV Serotype O and its Effects on the Immune Responses of Cattle

“…However, the recent studies showed that there is a ribosome skipping between 2A and 2B junction, which results in the production of P1 polyprotein of 80 kDa [11,33]. Along with 28 aa of L-protease the size of the fusion protein is 83 kDa which was the observed molecular weight.…”