Decoding the architecture of the varicella-zoster virus transcriptome

Abstract

SummaryVaricella-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, non-canonical ORFs, and ORF fusions, as well as putative non-coding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated as 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.

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

“…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 . Here, we performed more detailed dRNA-Seq analysis of the VLT and ORF63 loci in lytically VZV-infected human retina epithelial (ARPE-19) cells, identifying multiple lytic VLT ( lyt VLT) isoforms, all of which contain one or more upstream exons to the core VLT region (exons 1–5) that defines the latent isoform, referred to as VLT 7 .…”

“…Two major lyt VLT63 isoforms (i.e. lyt VLT63-1 and lyt VLT63-2) represent alternatively spliced variants of a lytic VLT isoform comprising the VLT core region, the additional upstream exon A, and the RNA 63-1 encoding canonical ORF63 15 . lyt VLT63-1 and lyt VLT63-2 differ from each other by skipping or retaining VLT exon 5, respectively.…”

“…lyt VLT63-1 and lyt VLT63-2 differ from each other by skipping or retaining VLT exon 5, respectively. Both isoforms use a splice acceptor site located 71 nucleotides (nt) upstream of the ORF63 coding sequence (CDS), located within the 5′-untranslated region (UTR) of canonical ORF63 15 – 17 . A third major isoform, lyt VLT63-3 utilizes a unique transcription start site (TSS), not used by any of the other lyt VLT and lyt VLT63 isoforms, proximal to exon 5 and is predicted to encode pORF63 (Fig.…”

Temporal Dynamics of HCMV Gene Expression in Lytic and Latent Infections

“…We discovered that in contrast to the traditional view of herpesvirus gene expression, the dependency of viral transcripts on metabolic drugs and viral transcripts' temporal dynamics are often decoupled, implying that expression kinetics and the dependency on protein synthesis or viral DNA replication are frequently two independent properties in viral gene expression regulation. A recent study on alpha herpesvirus, varicella-zoster virus (VZV), which mapped VZV transcripts also examined their expression kinetics and observed unexpected patterns of viral gene expression 46 . In addition to the traditional IE, Early and L kinetic classes, they defined groups of genes as Early-Late and Transactivated/True-Late (TA/TL) 46 .…”

“…A recent study on alpha herpesvirus, varicella-zoster virus (VZV), which mapped VZV transcripts also examined their expression kinetics and observed unexpected patterns of viral gene expression 46 . In addition to the traditional IE, Early and L kinetic classes, they defined groups of genes as Early-Late and Transactivated/True-Late (TA/TL) 46 . In their analysis, Early-Late genes show two waves of expression, one of which is dependent on de novo protein synthesis and a second, later wave of expression which depends on the onset of DNA-replication.…”