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

Depledge, Daniel P.; Mohr, Ian; Wilson, Angus C.

doi:10.1128/jvi.01342-18

Cited by 40 publications

(35 citation statements)

References 63 publications

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“…Standard methods for annotating viral transcriptomes require the integration of multiple types of Illumina RNA sequencing (RNA-Seq) data to identify transcription start sites (TSS), cleavage and polyadenylation sites (CPAS), splice sites, and transcript structures. The last is particularly challenging to infer using conventional short-read sequencing approaches (28). In contrast, direct RNA sequencing (dRNA-Seq) using Nanopore arrays offers the potential to capture all these distinct data points in a single sequencing run (10,29).…”

Section: Resultsmentioning

confidence: 99%

Decoding the Architecture of the Varicella-Zoster Virus Transcriptome

Braspenning

Sadaoka

Breuer

et al. 2020

mBio

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Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection. IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

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Section: Resultsmentioning

confidence: 99%

Decoding the Architecture of the Varicella-Zoster Virus Transcriptome

Braspenning

Sadaoka

Breuer

et al. 2020

mBio

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“…Sequencing of full-length RNAs, including direct RNA sequencing (dRNA-Seq), is particularly useful for disentangling complex loci at which multiple transcript isoforms overlap 13 , 14 . Recently, we used dRNA-Seq to reannotate the lytic VZV transcriptome and redefined the kinetic classes of all canonical and newly identified transcript isoforms 15 .…”

Section: Resultsmentioning

confidence: 99%

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

et al. 2020

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Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

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“…Sequencing of full length RNAs, including direct RNA sequencing dRNA Seq , is particularly useful for disentangling complex loci at which multiple RNA isoforms overlap 13,14 . dRNA Seq analysis of lytically VZV infected human retina epithelial ARPE cells identified numerous VLT isoforms, many of which utilized one or more major upstream exons, designated A, B, and C, upstream of the core VLT region exon that defines the latent isoform…”

Section: Ide Ifica I F L I Le Vlt Orf F I a C I I L Icall Vzv I Fec Ementioning

confidence: 99%

Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

Ouwendijk

Depledge

Rajbhandari

et al. 2020

Preprint

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SummaryVaricella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

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

Cited by 40 publications

References 63 publications

Decoding the Architecture of the Varicella-Zoster Virus Transcriptome

Decoding the Architecture of the Varicella-Zoster Virus Transcriptome

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

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