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...

Core promoter elements play a pivotal role in the transcriptional output, yet they are often detected manually within sequences of interest. Here, we present 2 contributions to the detection and curation of core promoter elements within given sequences. First, the Elements Navigation Tool (ElemeNT) is a user-friendly web-based, interactive tool for prediction and display of putative core promoter elements and their biologically-relevant combinations. Second, the CORE database summarizes ElemeNT-predicted core promoter elements near CAGE and RNA-seq-defined Drosophila melanogaster transcription start sites (TSSs). ElemeNT's predictions are based on biologically-functional core promoter elements, and can be used to infer core promoter compositions. ElemeNT does not assume prior knowledge of the actual TSS position, and can therefore assist in annotation of any given sequence. These resources, freely accessible at http://lifefaculty.biu.ac.il/gershon-tamar/index.php/resources, facilitate the identification of core promoter elements as active contributors to gene expression.

bPluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of "neurospheres," immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication. V aricella-zoster virus (VZV) replication is highly host restricted, growing efficiently only in human cells. In varicella, VZV typically infects and replicates in cutaneous fibroblasts and epidermal cells as well as several types of immune cells. VZV infections of central nervous system (CNS) vasculature are also not uncommonly observed, the virus infecting smooth muscle actinexpressing cells in vessel walls (16). VZV infects effectively primarily in a cell-associated manner in vitro, and it is thought that cell-to-cell spread occurs in most tissues. Cell-free virus is made in vivo by keratinocytes and is present in cutaneous vesicles (8), and released VZV appears to be an important component of T cell-toskin transmission in vivo (reviewed in reference 1).VZV infection of neurons is essential for establishment of latency and the ability to reactivate to cause herpes zoster. Initial neuronal infection by VZV is via cutaneous axons and retrograde transport to peripheral somatic and autonomic ganglia and/or by infected circulating lymphocytes that infiltrate the ganglia (28). VZV replicates in both neurons and ganglionic support cells of somatic and cranial peripheral sensory ganglia both upon initial infection and upon reactivation. Importantly, VZV causes a plethora of CNS diseases (due at least in part to infection of the vasculature) (16) and ocular diseases (reviewed in reference 9). The growth of VZV in neurons and the interactions that govern latency and reactivation have proven to be difficult to study outside the human host because of the species restriction of infection and the limited...

Varicella Zoster virus (VZV) productively infects humans causing varicella upon primary infection and herpes zoster upon reactivation from latency in neurons. In-vitro studies using cell-associated VZV infection have demonstrated productive VZV-infection, while a few recent studies of human neurons derived from stem cells incubated with cell-free, vaccine-derived VZV did not result in generation of infectious virus. In the present study, 90%-pure human embryonic stem cell-derived neurons were incubated with recombinant cell-free pOka-derived made with an improved method or with VZV vaccine. We found that cell-free pOka and vOka at higher multiplicities of infection elicited productive infection in neurons followed by spread of infection, cytopathic effect and release of infectious virus into the medium. These results further validate the use of this unlimited source of human neurons for studying unexplored aspects of VZV interaction with neurons such as entry, latency and reactivation.

The two human neurotropic alphaherpesviruses varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV1) both establish latency in sensory ganglia. Human trigeminal ganglia are known to frequently harbor both viruses, and there is evidence to suggest the presence of both VZV and HSV1 DNA in the same neuron. We ask here whether VZV and HSV1 can exclude themselves and each other and whether they can productively infect the same cells in human neurons and human foreskin fibroblasts (HFF). Simultaneous infection (coinfection) or consecutive infection (superinfection) was assessed using cell-free HSV1 and VZV expressing fluorescent reporter proteins. Automated analysis was carried out to detect singly and dually infected cells. We demonstrate that VZV and HSV1 both display efficient superinfection exclusion (SE) in HFF, with each virus excluding either itself or the other virus. While SE also occurred in neurons, it was with much lower efficiency. Both alphaherpesviruses productively infected the same neurons, whether applied simultaneously or even consecutively, albeit at lower frequencies. IMPORTANCESuperinfection exclusion by VZV for itself or the related neurotropic alphaherpesvirus HSV1 has been studied here for the first time. We find that while these viruses display classic SE in fibroblasts, SE is less efficient for both HSV1 and VZV in human neurons. The ability of multiple VZV strains to productively infect the same neurons has important implications in terms of recombination of both wild-type and vaccine strains in patients.