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

Werner J D Ouwendijk,Labchan Rajbhandari,Arun Venkatesan,Georges M G M Verjans,Tomohiko Sadaoka,Judith Breuer,Daniel P Depledge,Tihana Lenac Rovis,Stipan Jonjic

doi:10.1038/s41467-020-20031-4

Abstract

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.

Highlights

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans worldwide, reactivating in one-third to cause herpes zoster and occasionally chronic pain
During latency in human trigeminal ganglia (TG), VZV gene expression is restricted to transcripts arising from VLT (VZV latency-associated transcript) and ORF63 loci[7]
We apply nanopore direct RNA sequencing[11] to examine lytically VZV-infected epithelial cells and discover a novel set of VLT-ORF63 fusion transcripts, which are present in latently VZV-infected human TG and our recently improved in vitro VZV human neuronal latency model based on human induced pluripotent stem cell-derived sensory neurons (HSN)[7,12]

Summary

Introduction

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans worldwide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. While VLT is the most prevalent and abundant VZV transcript expressed in human TGs, lower levels of ORF63 RNA have been reported in up to 70% of examined latently VZV-infected ganglia[7,9]. This apparent expression of two distinct viral transcripts during latency is unique among wellstudied αHV. We apply nanopore direct RNA sequencing (dRNA-Seq)[11] to examine lytically VZV-infected epithelial cells and discover a novel set of VLT-ORF63 fusion transcripts, which are present in latently VZV-infected human TG and our recently improved in vitro VZV human neuronal latency model based on human induced pluripotent stem cell (iPSC)-derived sensory neurons (HSN)[7,12]

Methods

Results

Conclusion