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

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-as...

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Autores principales:	Shirley E. Braspenning, Robert Jan Lebbink, Daniel P. Depledge, Claudia M. E. Schapendonk, Laura A. Anderson, Georges M. G. M. Verjans, Tomohiko Sadaoka, Werner J. D. Ouwendijk
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	varicella-zoster virus VLT VLT-ORF63 CRISPR/Cas9 BAC mutagenesis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/96301de7e9954e899fc81bcb79e193cc
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spelling	oai:doaj.org-article:96301de7e9954e899fc81bcb79e193cc2021-11-25T19:14:13ZMutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection10.3390/v131122891999-4915https://doaj.org/article/96301de7e9954e899fc81bcb79e193cc2021-11-01T00:00:00Zhttps://www.mdpi.com/1999-4915/13/11/2289https://doaj.org/toc/1999-4915Primary 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.Shirley E. BraspenningRobert Jan LebbinkDaniel P. DepledgeClaudia M. E. SchapendonkLaura A. AndersonGeorges M. G. M. VerjansTomohiko SadaokaWerner J. D. OuwendijkMDPI AGarticlevaricella-zoster virusVLTVLT-ORF63CRISPR/Cas9BAC mutagenesisMicrobiologyQR1-502ENViruses, Vol 13, Iss 2289, p 2289 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	varicella-zoster virus VLT VLT-ORF63 CRISPR/Cas9 BAC mutagenesis Microbiology QR1-502
spellingShingle	varicella-zoster virus VLT VLT-ORF63 CRISPR/Cas9 BAC mutagenesis Microbiology QR1-502 Shirley E. Braspenning Robert Jan Lebbink Daniel P. Depledge Claudia M. E. Schapendonk Laura A. Anderson Georges M. G. M. Verjans Tomohiko Sadaoka Werner J. D. Ouwendijk Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
description	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.
format	article
author	Shirley E. Braspenning Robert Jan Lebbink Daniel P. Depledge Claudia M. E. Schapendonk Laura A. Anderson Georges M. G. M. Verjans Tomohiko Sadaoka Werner J. D. Ouwendijk
author_facet	Shirley E. Braspenning Robert Jan Lebbink Daniel P. Depledge Claudia M. E. Schapendonk Laura A. Anderson Georges M. G. M. Verjans Tomohiko Sadaoka Werner J. D. Ouwendijk
author_sort	Shirley E. Braspenning
title	Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
title_short	Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
title_full	Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
title_fullStr	Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
title_full_unstemmed	Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection
title_sort	mutagenesis of the varicella-zoster virus genome demonstrates that vlt and vlt-orf63 proteins are dispensable for lytic infection
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/96301de7e9954e899fc81bcb79e193cc
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Mutagenesis of the Varicella-Zoster Virus Genome Demonstrates That VLT and VLT-ORF63 Proteins Are Dispensable for Lytic Infection

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