Analysis of paramyxovirus transcription and replication by high-throughput sequencing

Wignall-Fleming, E. B., Hughes, D. J., Vattipally, S. , Modha, S. , Goodbourn, S., Davison, A. J. and Randall, R. E. (2019) Analysis of paramyxovirus transcription and replication by high-throughput sequencing. Journal of Virology, 93(17), e00571-19. (doi:10.1128/JVI.00571-19) (PMID:31189700) (PMCID:PMC6694822)

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Abstract

We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.

Item Type:Articles
Additional Information:This work was alsoe supported by the Wellcome Trust (grant nos. 101788/Z/13/Z, 101792/Z/13/Z and 109056/Z/15/A).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Wignall-fleming, Elizabeth and Davison, Professor Andrew and Randall, Professor Richard and Vattipally, Dr Sreenu and Modha, Ms Sejal
Authors: Wignall-Fleming, E. B., Hughes, D. J., Vattipally, S., Modha, S., Goodbourn, S., Davison, A. J., and Randall, R. E.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Virology
Publisher:American Society for Microbiology
ISSN:0022-538X
ISSN (Online):1098-5514
Published Online:12 June 2019
Copyright Holders:Copyright © 2019 Wignall-Fleming et al.
First Published:First published in Journal of Virology 93(17):e00571-19
Publisher Policy:Reproduced under a Creative Commons license

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
501443Centre for Integrated VirologyMassimo PalmariniMedical Research Council (MRC)G0801822MVLS III - CENTRE FOR VIRUS RESEARCH