Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

Nagel, Maria A.; Choe, Alexander; Traktinskiy, Igor; Cordery-Cotter, R.; Gilden, Don; Cohrs, Randall J.

doi:10.1128/jvi.01862-10

Cited by 58 publications

(72 citation statements)

References 34 publications

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“…We found that VZV-infected human neurons in cell culture transcribed every annotated ORF, unlike the limited viral transcription present in human and monkey ganglia latently infected with varicella-zoster virus (8)(9)(10). This confirmed a significant difference in virus gene transcription in vitro from that in human neurons latently infected with VZV.…”

supporting

confidence: 51%

Comparison of Varicella-Zoster Virus RNA Sequences in Human Neurons and Fibroblasts

Baird

Bowlin

Cohrs

et al. 2014

J Virol

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Varicella-zoster virus (VZV) infection causes varicella, after which the virus becomes latent in ganglionic neurons. In tissue culture, VZV-infected human neurons remain viable at 2 weeks, whereas fibroblasts develop cytopathology. Next-generation RNA sequencing was used to compare VZV transcriptomes in neurons and fibroblasts and identified only 12 differentially transcribed genes of the 70 annotated VZV open reading frames (ORFs), suggesting that defective virus transcription does not account for the lack of cell death in VZV-infected neurons in vitro.V aricella-zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus that usually produces varicella (chickenpox) upon primary infection, after which the virus becomes latent in ganglionic neurons along the entire neuraxis. In tissue culture, VZV-infected neurons remain viable 2 weeks after infection, while all nonneuronal cells develop a cytopathic effect (CPE). Our earlier studies of VZV-infected neurons derived from induced pluripotent stem (iPS) cells revealed that neurons appeared healthy 2 weeks later, with no detectable infectious virus in the tissue culture medium; analysis of the neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication (1). Furthermore, ultrastructural examination revealed few complete virions and numerous aberrant viral particles (2). Together, these findings indicate that VZV is not latent in these neurons, despite the lack of CPE, and suggest a deficiency in replication and viral assembly in neurons during the productive infection. Thus, nextgeneration RNA sequencing (NextGen RNA-seq) was used to examine all annotated VZV transcripts in both cell types.Terminally differentiated human neurons derived from human-induced pluripotent stem cells (iCell; Cellular Dynamics International, Madison, WI) and human fetal lung (HFL) fibroblasts were infected at a multiplicity of infection (MOI) of 1 ϫ 10Ϫ3 cell-free vOka strain VZ virions (Zostavax; Merck, Whitehouse Station, NJ) as described previously (2). Total RNA was extracted from VZV-infected fibroblasts when ϳ80% CPE was reached and from VZV-infected neurons 14 days postinfection (dpi), at which time 5 to 10% of the neurons were infected. cDNA libraries were constructed for each sample (3 neuron, 3 fibroblast samples) using the Illumina TruSeq stranded-mRNA sample preparation kit (Illumina, San Diego, CA). The six independently and uniquely indexed libraries were pooled and loaded onto a single lane of a HiSeq2000 flow cell for paired-end, 100-bp DNA sequencing using an Illumina HiSeq2000.NextGen RNA-seq of six cDNA libraries yielded 29.2 to 76.8 million total reads per sample (Fig. 1A). Removal of low-quality FPKMs from all libraries were separated using principal-component analysis (PCA). Principal component 1 (PC1) separated samples by their largest variance and resulted in separation between cell types. PC2 separated samples by the next-largest variance, independent of the first, and result...

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supporting

confidence: 51%

Comparison of Varicella-Zoster Virus RNA Sequences in Human Neurons and Fibroblasts

Baird

Bowlin

Cohrs

et al. 2014

J Virol

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“…Finally, we evaluated whether patient cells exhibited impaired ability to control VZV replication. VZVinfected MeWo cells were cocultured with PBMCs from controls or each of the 4 patients, and we measured expression of viral Orf63, which is among the most abundant viral transcripts (36). Consistent with the impaired IFN responses to VZV by PBMCs from P1, P3, and P4, we observed that VZV infection in MeWo cells gave rise to significantly higher viral gene expression when cocultured with PBMCs from P1, P3, or P4.…”

Section: Induction Of Ifns By At-rich Dna In Human Pbmcs Is Pol Iii-dmentioning

confidence: 56%

Inborn errors in RNA polymerase III underlie severe varicella zoster virus infections

Ogunjimi¹,

Zhang²,

Sørensen³

et al. 2017

Journal of Clinical Investigation

134

121

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“…Both VZV 63 and VZV gE proteins were found exclusively in neurons, but not in nonneuronal cells. Importantly, transcripts corresponding to VZV ORFs 21, 29, 62, 63, and 68 (gE) are present in latently infected human ganglia (1,19). VZV gE is also present in vivo months after experimental infection of human dorsal root ganglia (22).…”

Section: Discussionmentioning

confidence: 99%

“…This is because increasing numbers of VZV transcripts are now being detected with more advanced technologies (19), and the exact extent of viral gene expression is still unknown in latently infected human ganglia. In parallel with our continuing studies of VZV latency in human ganglia, this promising neuronal model will be used to analyze the total degree of VZV transcription and translation and will also be used to attempt to reactivate virus from nonproductively infected neurons.…”

Section: Discussionmentioning

confidence: 99%

Varicella-Zoster Virus Infection of Differentiated Human Neural Stem Cells

Pugazhenthi

Nair

Velmurugan

et al. 2011

J Virol

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Primary varicella-zoster virus (VZV) infection in humans produces varicella (chickenpox),Varicella zoster virus (VZV) is a ubiquitous, exclusively human, and highly neurotropic herpesvirus. Primary infection causes varicella (chickenpox), during which VZV replicates in the cells of every visceral organ and the skin (12). VZV establishes lifelong latency in neurons of cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis (9,11,17). VZV reactivates in elderly and immunocompromised individuals to produce shingles (herpes zoster), frequently complicated by chronic pain (postherpetic neuralgia) (10) and other serious neurological and ocular disorders, such as VZV vasculopathy, myelopathy, and retinal necrosis.Analyses of latently infected human ganglia obtained postmortem within 24 h after death have provided useful information on the configuration and abundance of the VZV genome and the limited extent of viral gene expression (reviewed in reference 6). However, such studies are hampered by lack of sufficient tissue, RNA degradation postmortem, and possible virus reactivation after death. Because no satisfactory animal model exists to study the interaction of VZV with neurons, alternate models have been developed. For example, immunedeficient SCID mice have had human neural stem cells or human ganglia transplanted, followed by infection with VZV and examination of infected neurons (2, 21). VZV has been also shown to infect cultured neurons from guinea pig enteric ganglia (8). An in vitro model of cultured human neurons infected with VZV provides an opportunity to study the molecular events leading to the establishment of VZV latency and reactivation. Here, we developed a system in which human neural stem cells (NSCs) were induced to differentiate into neurons, infected with VZV, observed carefully, and analyzed for viral gene expression and for markers of apoptosis.

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Varicella-Zoster Virus Transcriptome in Latently Infected Human Ganglia

Cited by 58 publications

References 34 publications

Comparison of Varicella-Zoster Virus RNA Sequences in Human Neurons and Fibroblasts

Comparison of Varicella-Zoster Virus RNA Sequences in Human Neurons and Fibroblasts

Inborn errors in RNA polymerase III underlie severe varicella zoster virus infections

Varicella-Zoster Virus Infection of Differentiated Human Neural Stem Cells

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