Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

• Published in: Nature communications 2019-02, Vol.10 (1), p.754-13, Article 754
• Main Authors: Depledge, Daniel P., Srinivas, Kalanghad Puthankalam, Sadaoka, Tomohiko, Bready, Devin, Mori, Yasuko, Placantonakis, Dimitris G., Mohr, Ian, Wilson, Angus C.
• Format: Article
• Language: English
• Subjects: 13/1; 38/91; 49/39; 49/91; 631/1647/350/1058; 631/1647/514/1949; 631/326/596/1553; 631/337/2019; 82/29; 82/80; Cell Line; Cells, Cultured; Complexity; Datasets; Epithelial Cells - virology; Error correction & detection; Fibroblasts; Fibroblasts - virology; Gene expression; Gene sequencing; Genes, Viral - genetics; Genome, Viral - genetics; Genomes; Glycoproteins; Herpes simplex; Herpes viruses; Herpesvirus 1, Human - genetics; Herpesvirus 1, Human - physiology; Host-Pathogen Interactions; Humanities and Social Sciences; Humans; Infections; Low level; Medicine; multidisciplinary; Nanopores; Neurons - cytology; Neurons - virology; Pathogens; Polyadenylation; Porosity; Proteins; Reproducibility; Ribonucleic acid; RNA; RNA, Viral - genetics; Science; Science (multidisciplinary); Sequence Analysis, RNA - methods; Splicing; Transcription initiation; Transcriptome - genetics; Ubiquitin; Ubiquitin-protein ligase; Viral Proteins - genetics; Viruses
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-08734-9

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes. Here, Depledge et al. use nanopore arrays for direct RNA sequencing to profile the HSV-1 transcriptome in productively infected cells. Sequencing of individual RNAs reveals a highly complex viral transcriptome including mRNAs encoding new viral fusion proteins derived by read-through transcription.