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2A is an oligopeptide sequence mediating a ribosome ‘skipping’ effect, producing an apparent ‘cleavage’ of polyproteins. First identified and characterized in picornaviruses, ‘2A-like’ sequences are found in other mammalian viruses and a wide range of insect viruses. Databases were analysed using a motif conserved amongst 2A/2A-like sequences. The newly identified 2A-like sequences (30 aa) were inserted into a reporter polyprotein to determine their cleavage activity. Our analyses showed that these sequences fall into two categories. The majority mediated very high (complete) cleavage to separate proteins and a few sequences mediated cleavage with lower efficiency, generating appreciable levels of the uncleaved form. Phylogenetic analyses of 2A-like sequences and RNA-dependent RNA polymerases (RdRps) indicated multiple, independent, acquisitions of these sequences at different stages during virus evolution. Within a virus family, 2A sequences are (probably) homologous, but diverge due to other evolutionary pressures. Amongst different families, however, 2A/2A-like sequences appear to be homoplasic.

During co-translational protein import into the endoplasmic reticulum ribosomes are docked onto the translocon. This prevents inappropriate exposure of nascent chains to the cytosol and, conversely, cytosolic factors from gaining access to the nascent chain. We exploited this property of co-translational translocation to examine the mechanism of polypeptide cleavage by the 2A peptide of the foot-and-mouth disease virus. We find that the scission reaction is unaffected by placing 2A into a co-translationally targeted protein. Moreover, the portion of the polypeptide C-terminal to the cleavage site remains in the cytosol unless it contains its own signal sequence. The pattern of cleavage is consistent with the proposal that the 2A-mediated cleavage reaction occurs within the ribosome itself. In addition, our data indicate that the ribosome-translocon complex detects the break in the nascent chain and prevents any downstream protein lacking a signal sequence from gaining access to the endoplasmic reticulum.Positive-strand RNA viruses typically encode polyproteins that are cleaved by viral or host-encoded proteinases (proteolytic processing) to produce mature, individual proteins (reviewed in Refs. 1 and 2). Alternatively proteins may be generated by translational effects such as ribosomal frameshifting or read-through of "leaky" stop codons. Such programmed alterations of translation are not virus-specific but widespread (although rare) mechanisms of gene expression (reviewed in Refs. 3 and 4). In foot-and-mouth disease virus (FMDV) 1 and some other picornaviruses the oligopeptide (ϳ20 amino acid) 2A region of the polyprotein mediates cleavage at its own C terminus to release it from the 2B region. 2A is also active when placed between reporter proteins and, therefore, cleavage requires no viral (proteinase) sequences outside this short peptide (5). Similarly, no host proteinases are known that cleave the 2A/2B site.Scission of 2A-containing polyproteins requires the correct protein rather than mRNA sequence (6). However, synthetic peptides containing 2A and "2A-like" sequences from other viruses do not autoproteolyse (7). Furthermore, on translation in vitro the portion of a polyprotein N-terminal to 2A typically accumulates in excess over the C-terminal portion. This imbalance is not because of protein degradation or nonspecific transcription/translation termination (6, 8). Modeling of 2A and 2A-like sequences indicates that the majority of each peptide can form an amphipathic helix whereas the amino acids immediately preceding the "cleavage" site (-NPGsP-) form a tight turn (7). A co-translational model for the cleavage reaction has been proposed (6, 7) in which the conformation of 2A places strain on the peptidyltransferase center of the ribosome, repositioning the peptidyl(2A)-tRNA ester linkage. This steric effect prohibits nucleophilic attack by the incoming (prolyl)-tRNA amide nitrogen that normally creates the new peptide bond. Instead, the N-terminal product is released from the ribosome by hydrolysis o...

The 18aa 2A self-cleaving oligopeptide from foot-andmouth disease virus can be used for co-expression of multiple, discrete proteins from a single ORF. 2A mediates a co-translational cleavage at its own C-terminus and is proposed to manipulate the ribosome into skipping the synthesis of a specific peptide bond (producing a discontinuity in the peptide backbone), rather than being involved in proteolysis. To explore the utility of the system to target discrete processing products, selfprocessing polyproteins comprising fluorescent proteins flanking 2A were constructed, permutating both the type of signal sequence and the location within the polyprotein. A polyprotein comprising a protein bearing an N-terminal signal sequence, 2A, then a protein lacking any signal sequence was constructed. Interestingly, both proteins were translocated into the endoplasmic reticulum. Despite the discontinuity in the peptide backbone, the mammalian ribosome:translocon complex did not disassemble -the second protein (lacking any signal) 'slipstreamed' through the translocon formed by the first (signal-bearing) protein. These polyprotein systems provide a novel method of targeting proteins to different subcellular sites by transfection with a plasmid encoding a single ORF. The inclusion of a fluorescent reporter enables visualisation of expression levels, whilst inclusion of a selectable marker enables stable cell-lines to be established rapidly.