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.