Abstract
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.
Highlights
Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns
Recent studies have shown that host transcription and mRNA processing are extensively remodeled during herpes simplex virus type 1 (HSV-1) infection[15,16,17], and recent studies using cDNAbased short- and long-read sequencing technologies indicate that the HSV-1 transcriptome, like other herpesviruses[6,18], may be substantially more complex than previously recognized[19,20,21]
To evaluate the reproducibility of direct RNA sequencing using nanopore arrays, total RNA was prepared from two biological replicates of normal human dermal fibroblasts (NHDF) infected with HSV-1 GFP-Us11 strain Patton[23,24] for 18 h
Summary
Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Loss of information on transcript isoform diversity, including splice variants, is especially problematic[14] Despite these inherent difficulties, recent studies have shown that host transcription and mRNA processing are extensively remodeled during HSV-1 infection[15,16,17], and recent studies using cDNAbased short- and long-read sequencing technologies indicate that the HSV-1 transcriptome, like other herpesviruses[6,18], may be substantially more complex than previously recognized[19,20,21]. This study demonstrates the power of direct RNA-seq to annotate complex viral transcriptomes and to identify novel polyadenylated RNA isoforms that further expand the coding potential of gene-dense viral genomes
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