Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

Interferon Gamma Prolongs Survival of Varicella-Zoster Virus-Infected Human Neurons In Vitro.

Infection of human neurons in vitro with varicella-zoster virus (VZV) at a low multiplicity of infection does not result in a cytopathic effect (CPE) within 14 days postinfection (dpi), despite production of infectious virus. We showed that by 28 dpi a CPE ultimately developed in infected neurons and that interferon gamma inhibited not only the CPE but also VZV DNA accumulation, transcription, and virus production, thereby prolonging the life of VZV-infected neurons.

Varicella zoster virus DNA does not accumulate in infected human neurons.

Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. It is unclear why human neurons infected in vitro with VZV at low multiplicity of infection do not exhibit a cytopathic effect (CPE) even though all VZV genes are transcribed, VZV proteins from all kinetic classes are translated and minimal infectious virus is produced. Here, we show that the lack of VZV-induced CPE correlates with the low abundance of viral DNA.

Comparison of varicella-zoster virus RNA sequences in human neurons and fibroblasts.

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.

Aberrant virion assembly and limited glycoprotein C production in varicella-zoster virus-infected neurons.

Highly pure (>95%) terminally differentiated neurons derived from pluripotent stem cells appear healthy at 2 weeks after infection with varicella-zoster virus (VZV), and the cell culture medium contains no infectious virus. Analysis of the healthy-appearing neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication. Herein, we further characterized virus in these neuronal cells, focusing on (i) transcription and expression of late VZV glycoprotein C (gC) open reading frame 14 (ORF14) and (ii) ultrastructural features of virus particles in neurons. The analysis showed that gC was not expressed in most infected neurons and gC expression was markedly reduced in a minority of VZV-infected neurons. In contrast, expression of the early-late VZV gE glycoprotein (ORF68) was abundant. Transcript analysis also showed decreased gC transcription compared with gE. Examination of viral structure by high-resolution transmission electron microscopy revealed fewer viral particles than typically observed in cells productively infected with VZV. Furthermore, viral particles were more aberrant, in that most capsids in the nuclei lacked a dense core and most enveloped particles in the cytoplasm were light particles (envelopes without capsids). Together, these results suggest a considerable deficiency in late-phase replication and viral assembly during VZV infection of neurons in culture.

Varicella zoster virus infection of highly pure terminally differentiated human neurons.

In vitro analyses of varicella zoster virus (VZV) reactivation from latency in human ganglia have been hampered by the inability to isolate virus by explantation or cocultivation techniques. Furthermore, attempts to study interaction of VZV with neurons in experimentally infected ganglion cells in vitro have been impaired by the presence of nonneuronal cells, which become productively infected and destroy the cultures. We have developed an in vitro model of VZV infection in which highly pure (>95 %) terminally differentiated human neurons derived from pluripotent stem cells were infected with VZV. At 2 weeks post-infection, infected neurons appeared healthy compared to VZV-infected human fetal lung fibroblasts (HFLs), which developed a cytopathic effect (CPE) within 1 week. Tissue culture medium from VZV-infected neurons did not produce a CPE in uninfected HFLs and did not contain PCR-amplifiable VZV DNA, but cocultivation of infected neurons with uninfected HFLs did produce a CPE. The nonproductively infected neurons contained multiple regions of the VZV genome, as well as transcripts and proteins corresponding to VZV immediate-early, early, and late genes. No markers of the apoptotic caspase cascade were detected in healthy-appearing VZV-infected neurons. VZV infection of highly pure terminally differentiated human neurons provides a unique in vitro system to study the VZV-neuronal relationship and the potential to investigate mechanisms of VZV reactivation.