Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2022
2022
Historique:
received:
03
02
2022
accepted:
11
10
2022
entrez:
10
11
2022
pubmed:
11
11
2022
medline:
15
11
2022
Statut:
epublish
Résumé
To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.
Identifiants
pubmed: 36355791
doi: 10.1371/journal.pone.0276697
pii: PONE-D-22-03411
pmc: PMC9648745
doi:
Substances chimiques
RNA, Messenger
0
RNA, Viral
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0276697Subventions
Organisme : Wellcome Trust
ID : 200838/Z/16/Z
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/R022011/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M02542X/1
Pays : United Kingdom
Informations de copyright
Copyright: © 2022 Donovan-Banfield et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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