title: Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping.
abstract: 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.
authors.affiliation: School of Medicine, Biomolecular Sciences Building, University of St Andrews, St Andrews KY16 9ST, UK.
authors.forename: Garth M
authors.initials: GM
authors.lastname: Funston
authors.affiliation: School of Medicine, Biomolecular Sciences Building, University of St Andrews, St Andrews KY16 9ST, UK.
authors.forename: Susanna E
authors.initials: SE
authors.lastname: Kallioinen
authors.affiliation: School of Medicine, Biomolecular Sciences Building, University of St Andrews, St Andrews KY16 9ST, UK.
authors.forename: Pablo
authors.initials: P
authors.lastname: de Felipe
authors.affiliation: School of Biology, Biomolecular Sciences Building, University of St Andrews, St Andrews KY16 9ST, UK.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Ryan
authors.affiliation: School of Medicine, Biomolecular Sciences Building, University of St Andrews, St Andrews KY16 9ST, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Iggo
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D050504:Oncolytic Viruses / Q000378:metabolism*; D010849:Picornaviridae / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus