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Journal of Virology, December 2008, p. 11824-11836, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01078-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bioinformatic and Physical Characterizations of Genome-Scale Ordered RNA Structure in Mammalian RNA Viruses ^,

Matthew Davis,¹ Selena M. Sagan,² John P. Pezacki,² David J. Evans,³ and Peter Simmonds^1*

Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom,¹ Department of Biochemistry, Microbiology & Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada, and Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada,² Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom³

Received 22 May 2008/ Accepted 10 September 2008

By the analysis of thermodynamic RNA secondary structure predictions, we previously obtained evidence for evolutionarily conserved large-scale ordering of RNA virus genomes (P. Simmonds, A. Tuplin, and D. J. Evans, RNA 10:1337-1351, 2004). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G+C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist.

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* Corresponding author. Mailing address: Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom. Phone: 44 131 650 7927. Fax: 44 131 650 6511. E-mail: Peter.Simmonds{at}ed.ac.uk

Published ahead of print on 17 September 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, December 2008, p. 11824-11836, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01078-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.