Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination.
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.
Dept. of Biochemistry & Molecular Biology, 201 Althouse Lab, University Park, PA 16802, USA.
Andrew
A
Woodman
Dept. of Biochemistry & Molecular Biology, 201 Althouse Lab, University Park, PA 16802, USA.
Jamie J
JJ
Arnold
Dept. of Biochemistry & Molecular Biology, 201 Althouse Lab, University Park, PA 16802, USA.
Craig E
CE
Cameron
Biomedical Sciences Research Complex, North Haugh, University of St. Andrews, St. Andrews KY16 9ST, UK d.j.evans@st-andrews.ac.uk.
David J
DJ
Evans
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D004261:DNA Replication; D021901:DNA, Intergenic / Q000235:genetics; D004770:Enterovirus / Q000201:enzymology* / Q000235:genetics*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D013698:Templates, Genetic; D014779:Virus Replication
D021901:DNA, Intergenic; D009711:Nucleotides; D012324:RNA-Dependent RNA Polymerase