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.
Highlights
We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication
It is not possible to distinguish reads generated from mRNAs from those generated from antigenomes by directional sequencing, the proportion of antigenome reads cannot exceed that of the L gene extended over the whole genome (2.6% of mRNA/antigenome reads overall)
While directional sequencing of poly(A)-selected RNA and a bioinformatic protocol can be used to separate genome RNA data from mRNA/antigenome data, the method suffers from the disadvantage that high levels of quantifiable genome RNA copurified with the poly(A)-selected mRNA, presumably as a consequence of RNA hybridization
Summary
We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. 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. The family Paramyxoviridae belongs to the order Mononegavirales and is populated by a large number of vertebrate viruses, some of which cause diseases in humans, including mumps, measles, and respiratory infections (https://talk.ictvonline.org/ taxonomy/). Respiroviruses and morbilliviruses have IG regions that are conserved in length and sequence within the genome, whereas rubulaviruses possess jvi.asm.org 2
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