The fidelity of the virus polymerase influences the rate of genetic recombination between viruses coinfecting the same cell. We used cell-based and new, biochemically-defined, assays to demonstrate that the viral polymerase is necessary and sufficient for the strand-transfer event of RNA virus recombination. Furthermore, the fidelity of the polymerase is critical in determining the efficiency with which recombination occurs; low fidelity polymerases exhibit high recombination rates, and vice versa.
The paper is published in Nucleic Acids Research:
Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination
Andrew Woodman; Jamie J. Arnold; Craig E. Cameron; David J. Evans
Nucleic Acids Research 2016; doi: 10.1093/nar/gkw567
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.