Long-Read Sequencing of Human Cytomegalovirus Transcriptome Reveals RNA Isoforms Carrying Distinct Coding Potentials

Abstract

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.

Comprehensive Annotations of Human Herpesvirus 6A and 6B Genomes Reveal Novel and Conserved Genomic Features

“…Laborious follow-up molecular work revealed the transcriptional architecture of individual genomic loci, but for most HHV-6 genes annotations are still based on these initial in-silico ORF predictions. In recent years, major advances in high-throughput sequencing approaches have revealed that the transcriptome and translatome of herpesviruses are extremely complex, encompassing large numbers of overlapping transcripts, extensive splicing and many non-canonical translation products (Arias et al, 2014;Balázs et al, 2017;Bencun et al, 2018;Depledge et al, 2019;Gatherer et al, 2011;Kara et al, 2019;O'Grady et al, 2019O'Grady et al, , 2016Tombácz et al, 2017;Whisnant et al, 2019). Our own work in which we employed ribosome profiling and systematic transcript analysis to decipher HCMV genome complexity revealed a rich collection of coding sequences that include many viral short ORFs (sORFs), uORFs and alternative translation products that generate extensions or truncations of canonical proteins (Stern-Ginossar et al, 2012).…”

The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

“…which is encoded along with US28 as a polycistronic transcript (Balazs et al, 2017). Thus, to ensure 237 US27 and US29 mRNA expression are unaffected by altered pUS28 expression, we assessed each of 238 these transcripts following lytic infection of fibroblasts with BFXwt-GFP or BFXstopUS28, as well 239 as TB40/EmCherry or TB40/EmCherry-US28Δ.…”

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

“…While producing comparatively low numbers of reads when compared to that from Illumina sequencing, the generation of long sequence reads obviates the need to computationally stitch together sequence fragments in order to reconstruct the original transcripts. Long-read RNA-Seq has been used to catalog transcript variation through alternative splicing and to identify novel transcripts or transcript isoforms (41,42). More importantly, when combined with short-read RNA-Seq and/or variant approaches such as CAGE-Seq, it enables fine detailing of viral transcriptomes at a very high resolution (43)(44)(45).…”