The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

N J Atkinson; Jeroen Witteveldt; D. J. Evans; Peter Simmonds

doi:10.1093/nar/gku075

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

N J Atkinson, Jeroen Witteveldt, D. J. Evans, Peter Simmonds

Royal (Dick) School of Veterinary Studies

Research output: Contribution to journal › Article › peer-review

Abstract

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.

Original language	English
Pages (from-to)	4527-4545
Journal	Nucleic Acids Research
Volume	42
Issue number	7
Early online date	26 Jan 2014
DOIs	https://doi.org/10.1093/nar/gku075
Publication status	Published - 2014

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10.1093/nar/gku075

Nucl. Acids Res.-2014-Atkinson
The Author(s) 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Final published version, 13.8 MBLicence: Creative Commons: Attribution (CC-BY)

http://nar.oxfordjournals.org/content/early/2014/01/25/nar.gku075

Innate immunity and endemic diseases in livestock species
Collie, D. (Principal Investigator), Beard, P. (Co-investigator), Bishop, S. (Co-investigator), Bronsvoort, M. (Co-investigator), Burt, D. (Co-investigator), Fitzgerald, R. (Co-investigator), Freeman, T. (Co-investigator), Gally, D. (Co-investigator), Gill, A. (Co-investigator), Glass, E. (Co-investigator), Hocking, P. (Co-investigator), Hope, J. (Co-investigator), Hume, D. (Co-investigator), Kaiser, P. (Co-investigator), Mabbott, N. (Co-investigator), McLachlan, G. (Co-investigator), Morrison, L. (Co-investigator), Stevens, J. (Co-investigator), Stevens, M. (Co-investigator) & Watson, M. (Co-investigator)
Biotechnology and Biological Sciences Research Council
1/04/12 → 31/03/17
Project: Research

Cite this

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title = "The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication",

abstract = "Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.",

author = "Atkinson, \{N J\} and Jeroen Witteveldt and Evans, \{D. J.\} and Peter Simmonds",

note = "Wellcome Trust [WT087628MA]; Programme Grant from the MRC [G0401584]. Funding for open access charge: University of Edinburgh.",

year = "2014",

doi = "10.1093/nar/gku075",

language = "English",

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pages = "4527--4545",

journal = "Nucleic Acids Research",

issn = "0305-1048",

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The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. / Atkinson, N J ; Witteveldt, Jeroen; Evans, D. J. et al.
In: Nucleic Acids Research, Vol. 42, No. 7, 2014, p. 4527-4545.

Research output: Contribution to journal › Article › peer-review

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T1 - The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

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AU - Witteveldt, Jeroen

AU - Evans, D. J.

AU - Simmonds, Peter

N1 - Wellcome Trust [WT087628MA]; Programme Grant from the MRC [G0401584]. Funding for open access charge: University of Edinburgh.

PY - 2014

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AB - Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.

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JO - Nucleic Acids Research

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The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

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Innate immunity and endemic diseases in livestock species

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