Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus...

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic 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 sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P¹⁷, G¹⁸, or P¹⁹, which displayed little or no "cleavage" activity in vitro, were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an 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 cotranslational "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.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic 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. They also indicate 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.