Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection

Abstract

Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.The interactions of the human neurotrophic herpesvirus varicella-zoster virus (VZV) with neurons have proven difficult to study because the virus shows fairly strict human specificity, and small-animal models do not fully recapitulate human disease. In humans, primary VZV infection follows viral inhalation and subsequent systemic delivery to the deep dermis of the skin via hemopoietic cells. In the course of the resulting disease (chickenpox), VZV infects sensory and sympathetic ganglion neurons, where it establishes a long period of latency. The infection of neurons may take place in the ganglia by circulating VZV-infected lymphocytes, or by virus infecting cutaneous nerve endings being retrogradely transported in the axon to the neuronal somata, as is the case with herpes simplex virus (HSV). VZV reactivation often leads to herpes zoster (shingles), a disease that is frequently associated with severe, debilitating, and often long-lasting intractable pain (postherpetic neuralgia) that is more often than not refractory to therapy.Few model systems of neuronal VZV infection have been developed. Two in vitro models are VZV infection of dissociated human neurons and intact human fetal dorsal root ganglia (DRG) (8, 9, 10). These studies have shed some light on VZV-neuronal interactions, demonstrating, for example, that VZV exerts antiapoptotic activities in neurons in the short term (maximum, 5 days) and that, unlike infected fibroblasts, infectious VZV is released from neurons.A human fetal DRG-SCID mouse model (22, 29; reviewed in reference 30) has al...

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…These results are similar to quiescent HSV-1 in neurons obtained from adult murine trigeminal ganglia wherein RNA-seq data suggest transcription from multiple regions of the virus genome in addition to LAT (Harkness et al, 2014). As the neurons can be maintained in compartmented microfluidic chambers that orientate neurite outgrowth, infection of axons with fluorescently tagged virus permits direct viewing of retrograde transport to neuronal cell bodies (Markus et al, 2011;Grigoryan et al, 2012). These nearpure (w95 % bIII-tubulin expressing) neuronal cultures provide an excellent opportunity to observe the outcome of VZV infection, such as induction of host anti-apoptotic genes (Markus et al, 2014), although latency (long-term presence of VZV DNA with limited transcription) has not yet been obtained.…”

“…During primary infection, both viruses gain access to neurons most likely through retrograde transport from the site of cutaneous lesion (Topp et al, 1994). Whilst there is no conclusive evidence showing virus accessing ganglia via retrograde axonal transport from cutaneous lesions in humans, retrograde axonal transport is supported by infectious virus in cutaneous vesicles (Grose & Brunel, 1978), virus-infected neurons in the region (Chen et al, 2004), and HSV-1 and VZV retrograde axonal transport in vitro (Antinone & Smith, 2010;Markus et al, 2011). VZV is also thought to enter ganglia haematogenously.…”

Aberrant Virion Assembly and Limited Glycoprotein C Production in Varicella-Zoster Virus-Infected Neurons

“…Later studies in which VZV-infected human fetal fibroblasts were cocultivated with explanted human fetal dorsal root ganglia indicated that neurons were resistant to apoptosis (4). Recently, heterogeneous cultures derived from human embryonic stem cells that contained neurons were infected with VZV, but again productive infection ensued, presumably due to the presence of a significant proportion of nonneuronal cells (5). When differentiated human neural stem cells that were Ͼ90% neurons based on immunostaining were infected with VZV, no cytopathic effect developed, and VZV DNA, virus-specific transcripts, and proteins were detected in healthy-appearing neurons (6).…”

Varicella-Zoster Virus Glycoprotein I Is Essential for Spread in Dorsal Root Ganglia and Facilitates Axonal Localization of Structural Virion Components in Neuronal Cultures

“…Synaptophysin and chromogranin A puncta were still readily observed along the lengths of the axons within our study, illustrating that the functionality of TGN secretory pathways was at least partly retained. Although a model of VZV retrograde transport has recently been reported (22,32), a model assessing anterograde spread of virus still remains in developmental stages. Thus, development of more- assessed by dual-immunofluorescence staining.…”

“…VZV infections of differentiated human neural stem cells (19) and terminally differentiated neurons derived from induced pluripotent stem cells (20,21) have been reported as persistent models of VZV infection. In contrast, human embryonic stem cell (22) and differentiated human neuroblastoma (23)-derived neuronal cells induced productive VZV infection under the study conditions utilized. The differentiated SH-SY5Y neuroblastoma cell culture model previously developed by our laboratory may be particularly useful to assess the neurovirulence of VZV deletion strains or mutant strains during productive VZV infection (23).…”