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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

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Published on: June 16, 2011

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Morgan R Herod¹, Eleni-Anna Loundras¹, Joseph C Ward¹

¹School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

The Journal of General Virology

|October 4, 2015

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.

Insights

Researchers developed new tools for studying foot-and-mouth disease virus (FMDV) RNA replication. Random mutagenesis identified insertion sites for epitope tags and replication-defective mutants, aiding molecular interaction studies.

Area of Science:

Virology
Molecular Biology
Genetics

Background:

Studying foot-and-mouth disease virus (FMDV) RNA replication complex interactions is challenging due to limited reagents.
Random insertional mutagenesis can identify essential protein domains and tolerant insertion sites.
Insertion sites can be modified with epitope tags for detection.

Purpose of the Study:

To develop novel reagents for probing FMDV RNA replication complex interactions.
To create a library of FMDV with genetic insertions using random mutagenesis.
To adapt insertion sites for epitope tagging and identify replication-defective mutants.

Main Methods:

Random transposon-mediated mutagenesis was used to create 15 nt insertions in the FMDV nonstructural polyprotein.
Replicon-based assays were employed to isolate replication-competent and defective insertion mutants.
Replication-competent sites were adapted for epitope tag incorporation.
Replication-defective mutants were assessed for rescue via helper replicon co-transfection.

Main Results:

A library of FMDV insertions was generated.
Replication-competent insertion sites were successfully adapted for epitope tagging.
Replication-defective mutants were identified.
Trans-complementation rescue of some replication-defective mutants was demonstrated, revealing intergenomic interactions.

Conclusions:

Epitope-tagged FMDV non-structural proteins provide valuable tools for downstream assays.
Replication-defective insertions serve as novel tools for studying intergenomic trans-complementation.
These tools will advance the understanding of picornavirus replication complexes.

Read full analysis

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