Varicella-zoster virus ORF63 inhibits apoptosis of primary human neurons

Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOkaΔORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.     

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

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

Neurons of the sensory ganglia are the major site of varicella-zoster virus (VZV) latency and may undergo productive infection during reactivation. Although the VZV glycoprotein E/glycoprotein I (gE/gI) complex is known to be critical for neurovirulence, few studies have assessed the roles of these proteins during infection of dorsal root ganglia (DRG) due to the high human specificity of the virus. Here, we show that the VZV glycoprotein I gene is an important neurotropic gene responsible for mediating the spread of virus in neuronal cultures and explanted DRG. Inoculation of differentiated SH-SY5Y neuronal cell cultures with a VZV gI gene deletion strain (VZV rOkaΔgI) showed a large reduction in the percentage of cells infected and significantly smaller plaque sizes in a comparison with cultures infected with the parental strain (VZV rOka). In contrast, VZV rOkaΔgI was not significantly attenuated in fibroblast cultures, demonstrating a cell type-specific role for VZV gI. Analysis of rOkaΔgI protein localization by immunofluorescent staining revealed aberrant localization of viral glycoprotein and capsid proteins, with little or no staining present in the axons of differentiated SH-SY5Y cells infected with rOkaΔgI, yet axonal vesicle trafficking was not impaired. Further studies utilizing explanted human DRG indicated that VZV gI is required for the spread of virus within DRG. These data demonstrate a role for VZV gI in the cell-to-cell spread of virus during productive replication in neuronal cells and a role in facilitating the access of virion components to axons.     

Three-Dimensional Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent Varicella-Zoster Virus Infection

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.     

Varicella-zoster virus infects human embryonic stem cell-derived neurons and neurospheres but not pluripotent embryonic stem cells or early progenitors

Pluripotent 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.     

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.     

Varicella-zoster virus infection of human foreskin fibroblast cells results in atypical cyclin expression and cyclin-dependent kinase activity

In its course of human infection, varicella-zoster virus (VZV) infects rarely dividing cells such as dermal fibroblasts, differentiated keratinocytes, mature T cells, and neurons, none of which are actively synthesizing DNA; however, VZV is able to productively infect them and use their machinery to replicate the viral genome. We hypothesized that VZV alters the intracellular environment to favor viral replication by dysregulating cell cycle proteins and kinases. Cyclin-dependent kinases (CDKs) and cyclins displayed a highly unusual profile in VZV-infected confluent fibroblasts: total amounts of CDK1, CDK2, cyclin B1, cyclin D3, and cyclin A protein increased, and kinase activities of CDK2, CDK4, and cyclin B1 were strongly and simultaneously induced. Cyclins B1 and D3 increased as early as 24 h after infection, concurrent with VZV protein synthesis. Confocal microscopy indicated that cyclin D3 overexpression was limited to areas of IE62 production, whereas cyclin B1 expression was irregular across the VZV plaque. Downstream substrates of CDKs, including pRb, p107, and GM130, did not show phosphorylation by immunoblotting, and p21 and p27 protein levels were increased following infection. Finally, although the complement of cyclin expression and high CDK activity indicated a progression through the S and G(2) phases of the cell cycle, DNA staining and flow cytometry indicated a possible G(1)/S blockade in infected cells. These data support earlier studies showing that pharmacological CDK inhibitors can inhibit VZV replication in cultured cells.     

Nuclear import of the varicella-zoster virus latency-associated protein ORF63 in primary neurons requires expression of the lytic protein ORF61 and occurs in a proteasome-dependent manner

Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shown to be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.     

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

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.