Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection.

Shirley E Braspenning,Laura A Anderson,Tomohiko Sadaoka,Robert Jan Lebbink,Daniel P Depledge,Werner J D Ouwendijk,Georges M G M Verjans,Claudia M E Schapendonk

doi:10.3390/v13112289

Abstract

Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells.

Highlights

Varicella-zoster virus (VZV) is a widespread human alphaherpesvirus and the causative agent of both varicella and herpes zoster (HZ or shingles) [1]
Since VZV latency-associated transcript (VLT) and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and bacterial artificial chromosome (BAC) technologies
VZV latency in sensory neurons is defined by selective expression of VLT and VLTORF63 fusion transcripts [9,10]

Summary

Introduction

Varicella-zoster virus (VZV) is a widespread human alphaherpesvirus and the causative agent of both varicella (chickenpox) and herpes zoster (HZ or shingles) [1]. In one-third of infected individuals latent VZV will reactivate to cause HZ later in life, a condition often complicated by chronic pain (post-herpetic neuralgia) for which currently no effective treatment is available [4,5]. During lytic infection VZV expresses at least 136 distinct polyadenylated RNAs, both coding and non-coding, that are expressed in a coordinated cascade [7,8]. VZV latency is characterized by expression of the VZV latency-associated transcript (VLT) and, to a lesser extent, low-level expression of two fusion transcripts of VLT and RNA 63 (VLT63-1 and VLT63-2) [9,10]. Unlike the single VLT isoform and two VLT63 isoforms expressed during latency, numerous lytic VLT (lytVLT) and VLT63 (lytVLT63) isoforms are produced by the usage of alternative transcription start-sites, read-through transcription and alternative splicing. The function of pVLT and pVLT-ORF63 during lytic VZV infection remains elusive

Methods

Results

Conclusion