The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences

J Gen Virol. 2001 May;82(Pt 5):1027-1041. doi: 10.1099/0022-1317-82-5-1027.


The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aphthovirus / genetics
  • Aphthovirus / metabolism*
  • Base Sequence
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • DNA, Viral
  • Glucuronidase / genetics
  • Green Fluorescent Proteins
  • Insect Viruses / genetics
  • Insect Viruses / metabolism
  • Luminescent Proteins / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Rotavirus / genetics
  • Rotavirus / metabolism
  • Trypanosoma / genetics
  • Trypanosoma / metabolism
  • Viral Proteins / biosynthesis*
  • Viral Proteins / genetics


  • DNA, Viral
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Viral Proteins
  • virus protein 2A
  • Green Fluorescent Proteins
  • Glucuronidase
  • Cysteine Endopeptidases
  • picornain 2A, Picornavirus