The architecture of the simian varicella virus transcriptome

Abstract

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed nanopore direct RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Comparative Analysis of the Simian Varicella Virus and Varicella Zoster Virus Genomes

“…Most recently, RNA sequencing approaches (RNA-Seq, dRNA-Seq) have provided comprehensive analyses of the SVV and VZV transcriptomes [ 32 , 33 ]. The transcriptional start sites (TSS) and cleavage and polyadenylation sites (CPAS) for each of the SVV and VZV protein coding genes have been identified.…”

“…RNA-Seq analysis has also provided a detailed characterization of the SVV VLT [ 32 ]. Similar to the VZV VLT, during lytic infection of culture cells, the SVV VLT locus is expressed as multiple spliced transcript isoforms with alternative transcription start sites.…”

“…However, in ganglia derived from latently infected monkeys, a single spliced VLT isoform was detected at low abundance. This VLT was detected within neurons but not non-neuronal cells of latently infected ganglia [ 32 ].…”

“…Expression of the SVV and VZV VLT oriented antisense to the ORF 61 and HSV-1 ICP0 homolog is consistent with gene expression during latency of other alphaherpesviruses [ 52 ]. HSV-1, HSV-2, EHV-1, EHV-4, PRV, bovine herpesvirus 1 (BHV-1), and feline herpesvirus 1 (FHV-1) each express a transcript antisense to an ICP0 homolog in neural ganglia of latently infected animals [ 32 ].…”

“…Multiple other isoforms are also transcribed from this locus during lytic infection. From Braspenning et al [ 32 ].…”