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A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61.

ABSTRACT: Varicella-zoster virus (VZV), an alphaherpesvirus, establishes lifelong latent infection in the neurons of >90% humans worldwide, reactivating in one-third to cause shingles, debilitating pain and stroke. How VZV maintains latency remains unclear. Here, using ultra-deep virus-enriched RNA sequencing of latently infected human trigeminal ganglia (TG), we demonstrate the consistent expression of a spliced VZV mRNA, antisense to VZV open reading frame 61 (ORF61). The spliced VZV latency-associated transcript (VLT) is expressed in human TG neurons and encodes a protein with late kinetics in productively infected cells in vitro and in shingles skin lesions. Whereas multiple alternatively spliced VLT isoforms (VLTly) are expressed during lytic infection, a single unique VLT isoform, which specifically suppresses ORF61 gene expression in co-transfected cells, predominates in latently VZV-infected human TG. The discovery of VLT links VZV with the other better characterized human and animal neurotropic alphaherpesviruses and provides insights into VZV latency.

SUBMITTER: Depledge DP

PROVIDER: S-EPMC5862956 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61.

Depledge Daniel P DP Ouwendijk Werner J D WJD Sadaoka Tomohiko T Braspenning Shirley E SE Mori Yasuko Y Cohrs Randall J RJ Verjans Georges M G M GMGM Breuer Judith J

Nature communications 20180321 1

Varicella-zoster virus (VZV), an alphaherpesvirus, establishes lifelong latent infection in the neurons of >90% humans worldwide, reactivating in one-third to cause shingles, debilitating pain and stroke. How VZV maintains latency remains unclear. Here, using ultra-deep virus-enriched RNA sequencing of latently infected human trigeminal ganglia (TG), we demonstrate the consistent expression of a spliced VZV mRNA, antisense to VZV open reading frame 61 (ORF61). The spliced VZV latency-associated ...[more]

PMID: 29563516

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Project description:The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). Next-generation sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis and therefore benefit from independent confirmation of the results. The antisense-to-latency transcript (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al., J Virol 86:7934-7942, 2010, https://doi.org/10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid capture assay and then employed transcriptome sequencing and novel reverse transcription-PCR (RT-PCR) assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at positions 120739/121012 and encodes a single splice site, which is shared with the 3'-coterminal K14-vGPCR/ORF74 mRNA, terminating at 130873 (GenBank accession number GQ994935), resulting in an ∼10,000-nucleotide transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5' untranslated region using a splice acceptor at 127888. In summary, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis. Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and lncRNAs, which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. The manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to monitor lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral miRNAs and thus has the potential to regulate this key part of the viral life cycle.

Project description:BackgroundThe inference of splicing orthology relationships between gene transcripts is a basic step for the prediction of transcripts and the annotation of gene structures in genomes. The splicing structure of a sequence refers to the exon extremity information in a CDS or the exon-intron extremity information in a gene sequence. Splicing orthologous CDS are pairs of CDS with similar sequences and conserved splicing structures from orthologous genes. Spliced alignment that consists in aligning a spliced cDNA sequence against an unspliced genomic sequence, constitutes a promising, yet unexplored approach for the identification of splicing orthology relationships. Existing spliced alignment algorithms do not exploit the information on the splicing structure of the input sequences, namely the exon structure of the cDNA sequence and the exon-intron structure of the genomic sequences. Yet, this information is often available for coding DNA sequences (CDS) and gene sequences annotated in databases, and it can help improve the accuracy of the computed spliced alignments. To address this issue, we introduce a new spliced alignment problem and a method called SplicedFamAlign (SFA) for computing the alignment of a spliced CDS against a gene sequence while accounting for the splicing structures of the input sequences, and then the inference of transcript splicing orthology groups in a gene family based on spliced alignments.ResultsThe experimental results show that SFA outperforms existing spliced alignment methods in terms of accuracy and execution time for CDS-to-gene alignment. We also show that the performance of SFA remains high for various levels of sequence similarity between input sequences, thanks to accounting for the splicing structure of the input sequences. It is important to notice that unlike all current spliced alignment methods that are meant for cDNA-to-genome alignments and can be used for CDS-to-gene alignments, SFA is the first method specifically designed for CDS-to-gene alignments.ConclusionWe show the usefulness of SFA for the comparison of genes and transcripts within a gene family for the purpose of analyzing splicing orthologies. It can also be used for gene structure annotation and alternative splicing analyses. SplicedFamAlign was implemented in Python. Source code is freely available at https://github.com/UdeS-CoBIUS/SpliceFamAlign .

Dataset Information

A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61.

Publications

A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene 61.

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