Modifications to the foot-and-mouth disease virus 2A peptide; influence on polyprotein processing and virus replication

Foot-and-mouth disease virus (FMDV) has a positive-sense ssRNA genome that includes a single, large, open reading frame encoding a polyprotein. The co-translational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a non-proteolytic mechanism termed "ribosome skipping" or "StopGo".

Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif (D(V/I)E(S/T)NPG↓P). The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing and virus viability were investigated. Amino acid substitutions are tolerated at residues E14, S15 and N16 within the 2A sequence of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30-50% compared to wt.

In contrast, no viruses were rescued containing substitutions at residues P17, G18 or P19 that displayed little or no "cleavage" activity in vitro, but wt revertants were obtained. The 2A substitutions impaired the replication of a FMDV replicon. Using transient expression assays, it was shown that certain amino acid substitutions at residues E14, S15, N16 and P19 resulted in partial "cleavage" of a protease-free polyprotein indicating that these specific residues are not essential for co-translational "cleavage".

Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV.ImportanceFoot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals.

Co-translational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a non-proteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA.

Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability and polyprotein processing. It also indicates that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV.