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Nucleic Acids Res. 2016 Aug 19;44(14):6883-95. doi: 10.1093/nar/gkw567. Epub 2016 Jun 17.

Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination.

Author information

1
Dept. of Biochemistry & Molecular Biology, 201 Althouse Lab, University Park, PA 16802, USA.
2
Biomedical Sciences Research Complex, North Haugh, University of St. Andrews, St. Andrews KY16 9ST, UK d.j.evans@st-andrews.ac.uk.

Abstract

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3' non-coding region we have further developed a cell-based assay (the 3'CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.

PMID:
27317698
PMCID:
PMC5001610
DOI:
10.1093/nar/gkw567
[Indexed for MEDLINE]
Free PMC Article

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