Herod, MR, Loundras, EA, Ward, J et al. (3 more authors) (2015) Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication. Journal of General Virology, 96 (12). pp. 3507-3518. ISSN 0022-1317
Abstract
Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2015, The Authors. This is an open access article published by the Microbiology Society under the Creative Commons Attribution License. |
Keywords: | FMDV; picornavirus; 3 A trans-complementation; transposon; mutagenesis |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) |
Funding Information: | Funder Grant number BBSRC 8804 (BB/K003801/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 12 Oct 2015 14:06 |
Last Modified: | 17 Jan 2018 05:16 |
Published Version: | http://dx.doi.org/10.1099/jgv.0.000306 |
Status: | Published |
Publisher: | Microbiology Society |
Identification Number: | 10.1099/jgv.0.000306 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:90710 |