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

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.

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

“…In the case of EBV, antiviral gene editing has targeted both the viral genome [90] as well as key cellular components required for EBV replication [93]. To our knowledge, CRISPR/Cas-mediated antiviral targeting has not yet been applied to VZV, although it has been used as a research tool to generate recombinant VZV [94].…”

Fraternal Twins: The Enigmatic Role of the Immune System in Alphaherpesvirus Pathogenesis and Latency and Its Impacts on Vaccine Efficacy

“…As noted earlier, older studies supported the notion that VZV latency was fundamentally different than that of HSV-1 and HSV-2 [ 39 ]. It has now been established that neurons latently infected with VZV do indeed liberally express a transcript (VLT) that is antisense to the ORF61 gene, an important transactivator and homolog to HSV ICP0 [ 7 ]. In addition to its apparent role in VZV latency, VLT is also expressed in some lytically infected cells [ 6 , 7 ].…”

“…It has now been established that neurons latently infected with VZV do indeed liberally express a transcript (VLT) that is antisense to the ORF61 gene, an important transactivator and homolog to HSV ICP0 [ 7 ]. In addition to its apparent role in VZV latency, VLT is also expressed in some lytically infected cells [ 6 , 7 ]. A microRNA (VZVsncRNA13) antisense to the leader sequence of ORF61 promotes VZV growth and spread in VZV-infected epithelial cells, possibly by inhibiting VLT [ 40 ].…”

“…As such, it is not possible to use these cells to study the establishment of latent VZV infection. This shortcoming could conceivably be overcome by using ganglionic tissue from aborted fetuses, since such cells would be unlikely to carry a latent infection [ 5 , 7 , 53 ]. However, given that the MRC-5 cell line, established more than half a century ago from the lung tissue of an aborted fetus and still under attack today by anti-vaccine and religious groups—an approach using human neuronal cell cultures from this source is certain to be deluged with ethical objections.…”

“…VZV latency was initially believed to differ substantially from HSV latency due to observations that as many as nine viral proteins were readily demonstrated in latently infected neurons [ 5 ]. The discovery of varicella latency transcript (VLT) implied that VZV latency probably more closely resembles that of HSV [ 6 , 7 ]. Whether or not any viral proteins are produced during VZV latency remains to be formally demonstrated.…”