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

The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated b y their 2A proteins 'deaving' at their own C termini, We have analysed this activity using artificial reporter polyprotein systems comprising green fl uorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A t o beta -glucuronidase (GUS) -forming a single, long, open reading frame. An alysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (similar to 90%) were in the form of the 'cleav age' products GUS and [GFP2A], Alternative models have been proposed to acc ount for the 'cleavage' activity: proteolysis by a host-cell proteinase, au toproteolysis or a translational effect. To investigate the mechanism of th is cleavage event constructs encoding site-directed mutant and naturally oc curring '2A-like' sequences were used to program in vitro translation syste ms and the gel profiles analysed. Analysis of site-directed mutant 2A seque nces showed that 'cleavage' occurred in constructs in which all the candida te nucleophilic residues were substituted - with the exception of aspartate -12, This residue is not, however, conserved amongst all functional '2A-lik e' sequences. '2A-like' sequences were identified within insect virus polyp roteins, the NS34 protein of type C rotaviruses, repeated sequences in Tryp anosoma spp, and a eubacterial alpha -glucosiduronase sequence (Thermatoga maritima aguA), All of the 2A-like sequences analysed were active (to vario us extents), other than the eubacterial alpha -glucosiduronase 2A-like sequ ence. This method of control of protein biogenesis may well not, therefore, be confined to members of the Picornaviridae, Taken together, these data p rovide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.