[Show abstract][Hide abstract] ABSTRACT: Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.
PLoS ONE 05/2015; 10(5):e0126081. DOI:10.1371/journal.pone.0126081 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retrograde axonal transport of the neurotropic alphaherpesvirus Varicella zoster virus (VZV) from vesicles at the skin results in sensory neuron infection and establishment of latency. Reactivation from latency leads to painful herpes zoster. The lack of a suitable animal model of these processes for the highly human-restricted VZV has resulted in a dearth of knowledge regarding the axonal transport of VZV. We recently demonstrated VZV infection of distal axons, leading to subsequent capsid transport to the neuronal somata, and replication and release of infectious virus using a new model based on neurons derived from human embryonic stem cells (hESC). In the present study, we perform a kinetic analysis of the retrograde transport of green fluorescent protein-tagged ORF23 in VZV capsids using hESC-derived neurons compartmentalized microfluidic chambers and time-lapse video microscopy. The motion of the VZV was discontinuous, showing net retrograde movement with numerous short pauses and reversals in direction. Velocities measured were higher 1 h after infection than 6 h after infection, while run lengths were similar at both time points. The hESC-derived neuron model was also used to show that reduced neuronal spread by a VZV loss-of-function mutant for ORF7 is not due to the prevention of axonal infection and transport of the virus to the neuronal somata. hESC-derived neurons are, therefore, a powerful model for studying axonal transport of VZV and molecular characteristics of neuronal infection.
Journal of NeuroVirology 08/2012; 18(6). DOI:10.1007/s13365-012-0124-z · 2.60 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of Virology 07/2011; 85(13):6220-33. DOI:10.1128/JVI.02396-10 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Schwann cells (SC), the glial cells of peripheral nerves, are involved in many diseases including Charcot Marie Tooth and neurofibromatosis, and play a pivotal role in peripheral nerve regeneration. Although it is possible to obtain human SC from nerve biopsies, they are difficult to maintain and expand in culture. Here we describe an efficient system for directing the differentiation of human embryonic stem cells (hESC) into cells with the morphological and molecular characteristics of SC. Neurospheres were generated from hESC using stromal cell induction and grown under conditions supportive of SC differentiation. After 8 weeks, hESC-derived SC expressed characteristic markers GFAP, S100, HNK1, P75, MBP and PMP-22, and were observed in close association with hESC-derived neurites. ~60% of the cells were double-immunostained for the SC markers GFAP/S100. RT-PCR analysis confirmed the expression of GFAP, S100, P75, PMP-22 and MBP and demonstrated expression of the SC markers P0, KROX20 and PLP in the cultures. Expression of CAD19 was observed in 2 and 4 week cultures and then was down-regulated, consistent with its expression in SC precursor, but not mature stages. Co-culture of hESC-derived SC with rat, chick or hESC-derived axons in compartmentalized microfluidic chambers resulted in tight association of the SC with axons. Apparent wrapping of the axons by SC was occasionally observed, suggestive of myelination. Our method for generating SC from hESC makes available a virtually unlimited source of human SC for studies of their role in nerve regeneration and modeling of disease.