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. 2019 Aug 13;93(17):e00571-19.
doi: 10.1128/JVI.00571-19. Print 2019 Sep 1.

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing

Elizabeth B Wignall-Fleming  1   2 , David J Hughes  1 , Sreenu Vattipally  2 , Sejal Modha  2 , Steve Goodbourn  3 , Andrew J Davison  2 , Richard E Randall  4
Affiliations

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing

Elizabeth B Wignall-Fleming et al. J Virol. .

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.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

Keywords: high-throughput sequencing; paramyxovirus; replication; transcription.

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Figures

FIG 1
FIG 1
Optimization of a workflow to study PIV5-W3 transcription and replication by nondirectional analysis of HTS data followed by directional analysis to distinguish mRNA/antigenome reads from genome reads. In panels a and b, colored boxes indicate approximate gene positions and contain the names of the genes. The individual colored vertical bars represent the coverage depth (number of reads) at each nucleotide in the reference sequence. (a) BWA alignments of the PIV5-W3 transcriptome in HSFs at 18 h p.i. analyzed using poly(A)-selected RNA and visualized in Tablet. (b and c) Comparison of mRNA/antigenome and genome RNA abundance relative to total RNA after poly(A) selection or rRNA reduction of total cell RNA. RNA was extracted from PIV5-W3-infected A549 cells at 6, 12, and 18 h p.i., and the reads were subjected to directional analysis. (b) BWA alignments for mRNA/antigenome and genome reads at 18 h p.i. visualized in Tablet. (c) Abundance of mRNA/antigenome and genome reads at 6, 12, and 18 h p.i.
FIG 2
FIG 2
Kinetic analysis of PIV2-Co, PIV3-Wash, PIV5-W3, and MuV-Enders transcription and replication. The relative abundances of mRNA and genome reads were compared to the number of total reads at various times p.i. A549 cells were infected at an MOI of 10 to 20 PFU per cell, and total RNA was isolated at various times p.i. Following physical removal of rRNA and mitochondrial RNA, the samples were subjected to library preparation, sequencing, and directional analysis, followed by bioinformatical removal of residual rRNA and mitochondrial reads. The bars show SD values based on three experiments.
FIG 3
FIG 3
Comparison of the mRNA abundance gradients of PIV2-Co, PIV3-Wash, PIV5-W3, and MuV-Enders with time p.i. The RNA samples described for Fig. 2 were subjected to bioinformatic analysis to determine the percent contribution of individual viral mRNAs to the total viral mRNA population.
FIG 4
FIG 4
Analysis of RNA editing. Shown are relative abundances of the P, V, and I mRNAs for PIV2-Co, PIV5-W3, and MuV-Enders (orthorubulaviruses) and the P, V, and D mRNAs for PIV3-Wash (respiroviruses) in the RNA samples described for Fig. 2. The number of reads generated from the RNA editing site was calculated using a 10-nt search string immediately upstream and downstream of the site. The number of inserted G residues in the reads overlapping the RNA editing site that generated the V, P, and I mRNA transcripts was calculated, 0 and 0 + 3 G inserts (V or P for orthorubulaviruses and respiroviruses, respectively), 2 and 2 + 3 G inserts (P or D for orthorubulaviruses and respiroviruses, respectively), and 1 and 1 + 3 G inserts (I or V for orthorubulaviruses or respiroviruses, respectively). The bars show SD values based on three independent experiments.
FIG 5
FIG 5
Relative abundance of readthrough mRNAs compared to the average coverage of the gene immediately upstream for PIV2-Co, PIV5-W3, MuV-Enders, and PIV3-Wash. The average coverage of reads overlapping the IG was compared to the average coverage read depth of the gene immediately upstream of the IG region. The bars show SD values based on three independent experiments.
FIG 6
FIG 6
Effects of strain differences on PIV5 transcription and replication. (a) The relative abundance of PIV5-CPI+ mRNA and genome reads were compared to the number of total reads at various times p.i. in A549 cells. Total RNA was isolated and, following physical removal of rRNA and mitochondrial RNA, was subjected to library preparation, HTS, and directional read analysis, followed by bioinformatic removal of residual rRNA and mitochondrial RNA sequences. The mRNA abundance gradient (b), the relative abundance of the P, V, and I mRNAs (c), and the generation of readthrough mRNAs (d) were determined from the data sets as described for Fig. 3 to 5, respectively.
FIG 7
FIG 7
Transcriptional and replicative differences of PIV5 recombinant virus rPIV5-W3:P(F157) (replacement of the serine residue at position 157 by a phenylalanine residue). (a) The relative abundance of rPIV5-W3:P(F157) mRNA and genome reads were compared to the number of total reads at 24 h p.i. A549 cells were infected at an MOI of 10 to 20 PFU/cell and total cell RNA was isolated at various times p.i. rRNA and mitochondrial RNA were physically removed and the RNA was subjected to library preparation, sequencing, and directional analysis, followed by bioinformatic removal of residual rRNA and mitochondrial RNA sequences. The mRNA abundance gradient (b), the relative abundance of the P, V, and I mRNAs (c), and the generation of readthrough mRNAs (d) were determined from the data sets as described for Fig. 3 to 5, respectively.
FIG 8
FIG 8
Theoretical mRNA abundance gradients compared to actual gradients in a model in which vRdRP disengages with equal chance at any nucleotide during transcription, and truncated, non-poly(A) mRNAs are rapidly degraded. (a) Model of the relative abundance of individual viral mRNAs in which position 1 of the genome constitutes 100% of transcripts and the last nucleotide constitutes 1 to 2%. The end of each gene is indicated where polyadenylation occurs at the U tract to generate mRNAs that are subsequently translated. In this model it is assumed that transcripts that are prematurely terminated when vRdRP disengages from the genome upstream of the U tract are not polyadenylated and are degraded rapidly. The stepwise transcription profiles therefore reflect the theoretical abundance of polyadenylated mRNAs. (b) The theoretical percentage contribution of polyadenylated viral mRNAs to the total viral mRNA population, as calculated from the theoretical gradient shown in panel a. (c) The mRNA abundance gradient determined experimentally for cells infected with PIV2-Co, PIV5-W3, MuV, or PIV3-Wash at 12 h p.i. as described for Fig. 2.
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