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

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2289
Author(s):  
Shirley E. Braspenning ◽  
Robert Jan Lebbink ◽  
Daniel P. Depledge ◽  
Claudia M. E. Schapendonk ◽  
Laura A. Anderson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Varicella Zoster Virus ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Lytic Infection ◽  
Viral Gene ◽  
Growth Curve Analysis ◽  
Potential Growth ◽  
Varicella Zoster ◽  
Ganglion Neurons

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.

scholarly journals Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

2020 ◽  
Author(s):  
Werner J. D. Ouwendijk ◽  
Daniel P. Depledge ◽  
Labchan Rajbhandari ◽  
Tihana Lenac Rovis ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
Viral Gene Expression ◽  
Fusion Transcript ◽  
Viral Gene ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Rapid Amplification

SummaryVaricella-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. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. 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.

scholarly journals Varicella Zoster Virus in the Nervous System

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1356 ◽  
Author(s):  
Don Gilden ◽  
Maria Nagel ◽  
Randall Cohrs ◽  
Ravi Mahalingam ◽  
Nicholas Baird
Keyword(s):  
Nervous System ◽  
Varicella Zoster Virus ◽  
Clinical Laboratory ◽  
Viral Gene Expression ◽  
Neurological Complications ◽  
Viral Gene ◽  
Cell Mediated Immunity ◽  
Specific Cell ◽  
Varicella Zoster ◽  
Cranial Nerve Palsies

Varicella zoster virus (VZV) is a ubiquitous, exclusively human alphaherpesvirus. Primary infection usually results in varicella (chickenpox), after which VZV becomes latent in ganglionic neurons along the entire neuraxis. As VZV-specific cell-mediated immunity declines in elderly and immunocompromised individuals, VZV reactivates and causes herpes zoster (shingles), frequently complicated by postherpetic neuralgia. VZV reactivation also produces multiple serious neurological and ocular diseases, such as cranial nerve palsies, meningoencephalitis, myelopathy, and VZV vasculopathy, including giant cell arteritis, with or without associated rash. Herein, we review the clinical, laboratory, imaging, and pathological features of neurological complications of VZV reactivation as well as diagnostic tests to verify VZV infection of the nervous system. Updates on the physical state of VZV DNA and viral gene expression in latently infected ganglia, neuronal, and primate models to study varicella pathogenesis and immunity are presented along with innovations in the immunization of elderly individuals to prevent VZV reactivation.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Werner J. D. Ouwendijk ◽  
Daniel P. Depledge ◽  
Labchan Rajbhandari ◽  
Tihana Lenac Rovis ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
World Wide ◽  
Viral Gene Expression ◽  
Fusion Transcript ◽  
Viral Gene ◽  
Trigeminal Ganglia ◽  
Varicella Zoster ◽  
Viral Gene Transcription

AbstractVaricella-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.

scholarly journals RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

2014 ◽  
Vol 10 (1) ◽  
pp. e1003896 ◽  
Author(s):  
Meleri Jones ◽  
Inga R. Dry ◽  
Dan Frampton ◽  
Manuraj Singh ◽  
Ravinder K. Kanda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
Viral Gene Expression ◽  
Keratinocyte Differentiation ◽  
Viral Gene ◽  
Rna Seq ◽  
Varicella Zoster

scholarly journals Varicella-Zoster Virus Gene Expression in Latently Infected and Explanted Human Ganglia

2000 ◽  
Vol 74 (24) ◽  
pp. 11893-11898 ◽  
Author(s):  
Peter G. E. Kennedy ◽  
Esther Grinfeld ◽  
Jeanne E. Bell
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
Human Subjects ◽  
Viral Gene Expression ◽  
Viral Reactivation ◽  
Hiv Positive ◽  
Viral Gene ◽  
Varicella Zoster ◽  
Latently Infected ◽  
Gene 4

ABSTRACT A consistent feature of varicella-zoster virus (VZV) latency is the restricted pattern of viral gene expression in human ganglionic tissues. To understand further the significance of this gene restriction, we used in situ hybridization (ISH) to detect the frequency of RNA expression for nine VZV genes in trigeminal ganglia (TG) from 35 human subjects, including 18 who were human immunodeficiency virus (HIV) positive. RNA for VZV gene 21 was detected in 7 of 11 normal and 6 of 10 HIV-positive subjects, RNA for gene 29 was detected in 5 of 14 normal and 11 of 11 HIV-positive subjects, RNA for gene 62 was detected in 4 of 10 normal and 6 of 9 HIV-positive subjects, and RNA for gene 63 was detected in 8 of 17 normal and 12 of 15 HIV-positive subjects. RNA for VZV gene 4 was detected in 2 of 13 normal and 4 of 9 HIV-positive subjects, and RNA for gene 18 was detected in 4 of 15 normal and 5 of 15 HIV-positive subjects. By contrast, RNAs for VZV genes 28, 40, and 61 were rarely or never detected. In addition, immunocytochemical analysis detected the presence of VZV gene 63-encoded protein in five normal and four HIV-positive subjects. VZV RNA was also analyzed in explanted fresh human TG and dorsal root ganglia from five normal human subjects over a period of up to 11 days in culture. We found a very different pattern of gene expression in these explants, with transcripts for VZV genes 18, 28, 29, 40, and 63 all frequently detected, presumably as a result of viral reactivation. Taken together, these data provide further support for the notion of significant and restricted viral gene expression in VZV latency.

scholarly journals Decoding the Architecture of the Varicella-Zoster Virus Transcriptome

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Shirley E. Braspenning ◽  
Tomohiko Sadaoka ◽  
Judith Breuer ◽  
Georges M. G. M. Verjans ◽  
Werner J. D. Ouwendijk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Zoster ◽  
Varicella Zoster Virus ◽  
Rna Sequencing ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Viral Gene ◽  
Varicella Zoster ◽  
Kinetic Class ◽  
Coding Potential

ABSTRACT Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life to cause herpes zoster, commonly associated with chronic pain. The VZV genome is densely packed and produces multitudes of overlapping transcripts deriving from both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we integrated multiple short-read RNA sequencing approaches with long-read direct RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs (ncRNAs). Furthermore, we determined the kinetic class of all VZV transcripts and observed, unexpectedly, that transcripts encoding the ORF62 protein, previously designated Immediate-Early, were expressed with Late kinetics. Our work showcases the complexity of the VZV transcriptome and provides a comprehensive resource that will facilitate future functional studies of coding RNAs, ncRNAs, and the biological mechanisms underlying the regulation of viral transcription and translation during lytic VZV infection. IMPORTANCE Transcription from herpesviral genomes, executed by the host RNA polymerase II and regulated by viral proteins, results in coordinated viral gene expression to efficiently produce infectious progeny. However, the complete coding potential and regulation of viral gene expression remain ill-defined for the human alphaherpesvirus varicella-zoster virus (VZV), causative agent of both varicella and herpes zoster. Here, we present a comprehensive overview of the VZV transcriptome and the kinetic class of all identified viral transcripts, using two virus strains and two biologically relevant cell types. Additionally, our data provide an overview of how VZV diversifies its transcription from one of the smallest herpesviral genomes. Unexpectedly, the transcript encoding the major viral transactivator protein (pORF62) was expressed with Late kinetics, whereas orthologous transcripts in other alphaherpesviruses are typically expressed during the immediate early phase. Therefore, our work both establishes the architecture of the VZV transcriptome and provides insight into regulation of alphaherpesvirus gene expression.

scholarly journals Array Analysis of Viral Gene Transcription during Lytic Infection of Cells in Tissue Culture with Varicella-Zoster Virus

2003 ◽  
Vol 77 (21) ◽  
pp. 11718-11732 ◽  
Author(s):  
Randall J. Cohrs ◽  
Michael P. Hurley ◽  
Donald H. Gilden
Keyword(s):  
Varicella Zoster Virus ◽  
Relative Abundance ◽  
Gene Array ◽  
Viral Gene Expression ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Viral Gene ◽  
Array Analysis ◽  
Varicella Zoster ◽  
Infected Cells

ABSTRACT Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, causes childhood chickenpox (varicella), becomes latent in dorsal root and autonomic ganglia, and reactivates decades later to cause shingles (zoster) and other neurologic complications. Although the sequence and configuration of VZV DNA have been determined, relatively little is known about viral gene expression in productively infected cells. This is in part because VZV is highly cell associated, and sufficient titers of cell-free virus for use in synchronizing infection do not develop. PCR-based transcriptional arrays were constructed to simultaneously determine the relative abundance of the ≈70 predicted VZV open reading frames (ORFs). Fragments (250 to 600 bp) from the 5′ and 3′ end of each ORF were PCR amplified and inserted into plasmid vectors. The virus DNA inserts were amplified, quantitated, and spotted onto nylon membranes. Probing the arrays with radiolabeled cDNA synthesized from VZV-infected cells revealed an increase in the magnitude of the expressed VZV genes from days 1 to 3 after low-multiplicity virus infection but little change in their relative abundance. The most abundant VZV transcripts mapped to ORFs 9/9A, 64, 33/33A, and 49, of which only ORF 9 corresponded to a previously identified structural gene. Array analysis also mapped transcripts to three large intergenic regions previously thought to be transcriptionally silent, results subsequently confirmed by Northern blot and reverse transcription-PCR analysis. Array analysis provides a formidable tool to analyze transcription of an important ubiquitous human pathogen.

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