title: Employing transposon mutagenesis to investigate  foot-and-mouth disease virus replication.
abstract: Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be adapted by the incorporation of commonly used epitope tags, facilitating their detection with commercially available reagents. In this study, we used random transposon-mediated mutagenesis to produce a library of 15 nt insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay, we isolated multiple replication-competent as well as replication-defective insertions. We adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins for use in a variety of downstream assays. Additionally, we showed that replication of some of the replication-defective insertion mutants could be rescued by co-transfection of a ‘helper’ replicon, demonstrating a novel use of random mutagenesis to identify intergenomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornavirus replication complexes.
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Herod
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Eleni-Anna
authors.initials: EA
authors.lastname: Loundras
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Ward
authors.affiliation: Biomedical Sciences Research Complex (BSRC), School of Biology, University of St Andrews, North Haugh, St Andrews, KY16 9ST, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: Tulloch
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Stonehouse
mesh_terms: D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D004251:DNA Transposable Elements; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins