Summary auto-generated
This study investigates how the 2A proteins of aphthoviruses and cardioviruses mediate cleavage of viral polyproteins. The 2A region of foot-and-mouth disease virus (FMDV) is only 16-18 amino acids long, yet it can cleave efficiently (~85%) when inserted between two reporter proteins (CAT and GUS) in an artificial system. The C-terminal 18-19 amino acid regions of cardiovirus 2A proteins from Theiler's murine encephalomyelitis virus (TME) and encephalomyocarditis virus (EMC) also mediate cleavage with ~85% efficiency, comparable to FMDV 2A. Notably, the entire TME 2A protein (150 amino acids) cleaves nearly completely (~99%) when fused to GUS. Adding FMDV capsid protein 1D sequences upstream of the 2A region increases cleavage efficiency to ~99%. The cleavage mechanism requires eukaryotic translation machinery, as no cleavage was detected in prokaryotic systems. The highly conserved C-terminal sequence motif (NPG) and downstream proline residue appear critical for cleavage activity, suggesting a role in ribosomal or cellular recognition during translation.
Key findings
- The 16-18 amino acid 2A region of FMDV and the C-terminal 18-19 amino acids of cardiovirus 2A proteins mediate polyprotein cleavage with ~85% efficiency in artificial constructs
- Upstream FMDV capsid protein sequences significantly enhance 2A-mediated cleavage efficiency to ~99%, indicating the upstream context influences activity
- The conserved C-terminal sequence motif (-NPG-P-) is essential for cleavage, present in all tested aphtho- and cardiovirus 2A proteins
- 2A-mediated cleavage requires eukaryotic translation systems and does not occur in prokaryotic expression systems, suggesting a co-translational, ribosome-dependent mechanism
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Abstract
The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta- glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. 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 efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A- GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT- delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.