Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Abstract

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data

“…Some very possible, low-copy ORFs were added to form the final set of ORFs, which was used for transcript grouping and genome annotation. This strategy was different from those used for HAdV-C transcriptome analysis with deep sequencing data because HAdV-C has already been intensively studied and has a reliable transcriptome map for reference [ 4 , 10 , 13 ]. The strategy of de novo ORF prediction was reasonable and easy to carry out, which could be used to parse transcriptomes of novel adenoviruses in the future.…”

“…The life cycle of HAdV in host cells is artificially divided into early and late phases according to the onset of the replication of the viral genome [ 3 ]. Virus replication is an accurately programmed process that is regulated, implemented and manifested by the sequential expression of viral genes [ 4 , 5 ]. To compact large genetic information into a relatively small genome, adenovirus organizes viral genes as transcription units, and distributes them on both forward and reverse strands.…”

“…Generally, a transcription unit comprises a promoter, several viral open reading frames (ORFs), polyadenylation signals (PAS), as well as splicing donors and acceptors. The strategy is typically exhibited in the late transcription unit [ 4 , 6 , 7 ]. The major late promoter (MLP) of HAdV-5 controls the expression of virus late genes, which encode the proteins involved in virion assembly and all viral structural proteins except pIX.…”

“…In 2010, a promoter inside the L4 region was found to control the expression of 22 kD and 33 kD [ 12 ]. Recently, three groups combined next-generation sequencing with nanopore-long sequencing technologies to restudy the transcriptome of HAdV-5 or HAdV-2 [ 4 , 10 , 13 ]. Huge novel low-abundance transcripts were discovered and taken as the background evolution potential for adenoviruses to fit new environments.…”

“…Huge novel low-abundance transcripts were discovered and taken as the background evolution potential for adenoviruses to fit new environments. Price AM et al even found a fused protein of E4ORF6 and DBP in HAdV-5, which played an important role in the formation of the viral replication center [ 4 ].…”