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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 ha...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001610/ https://www.ncbi.nlm.nih.gov/pubmed/27317698 http://dx.doi.org/10.1093/nar/gkw567 |
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author | Woodman, Andrew Arnold, Jamie J. Cameron, Craig E. Evans, David J. |
author_facet | Woodman, Andrew Arnold, Jamie J. Cameron, Craig E. Evans, David J. |
author_sort | Woodman, Andrew |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5001610 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-50016102016-12-07 Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination Woodman, Andrew Arnold, Jamie J. Cameron, Craig E. Evans, David J. Nucleic Acids Res Nucleic Acid Enzymes 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. Oxford University Press 2016-08-19 2016-06-17 /pmc/articles/PMC5001610/ /pubmed/27317698 http://dx.doi.org/10.1093/nar/gkw567 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Nucleic Acid Enzymes Woodman, Andrew Arnold, Jamie J. Cameron, Craig E. Evans, David J. Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title | Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title_full | Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title_fullStr | Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title_full_unstemmed | Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title_short | Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
title_sort | biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001610/ https://www.ncbi.nlm.nih.gov/pubmed/27317698 http://dx.doi.org/10.1093/nar/gkw567 |
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