RESUMEN
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.
Asunto(s)
Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Genoma Viral , Infecciones por Picornaviridae/virología , Picornaviridae/genética , Poliproteínas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Biológica , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Picornaviridae/metabolismo , Poliproteínas/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Alineación de SecuenciaRESUMEN
Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX-2A-GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner.