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Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain., Author summary Acute and chronic pain are common complications of herpes zoster, but the mechanisms underlying the transition to chronic pain states remain poorly understood. Varicella-zoster virus (VZV)-inoculated rats develop persistent behaviors that indicate the development of prolonged hypersensitivity that may model postherpetic neuralgia (PHN) seen in the clinic. To address the requirements of viral gene expression and replication for the induction of pain, we developed mutant VZV and applied them to rat models of PHN. Our results reveal the production of essential VZV regulatory proteins is required to induce nocifensive behaviors, but that full productive virus replication is dispensable. These data suggest a mechanism for pain induction that involves the early expression of VZV regulatory proteins in abortively infected neurons after herpes zoster that may cause aberrant host pain signaling and PHN.

Summary Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4+ T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4+ cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease.

Pain is a common complication of herpes zoster (HZ) infection which results from reactivation of a latent varicella zoster virus (VZV). A third of HZ patients’ progress to a chronic pain state known as post herpetic neuralgia (PHN), and about a quarter of these patients’ have orofacial pain. The mechanisms controlling the pain responses are not understood. Studies suggest central pathways involving the thalamus could control pain related to HZ, and studies in our lab suggest vesicular GABA transporter (VGAT) in the lateral thalamus influences orofacial pain. We hypothesized that thalamic VGAT functions, in part, to reduce pain, particularly orofacial pain, associated with VZV. To address this hypothesis VZV was injected into the whisker pad. Affective and motivational aspects of pain were measured using the Place Escape/Avoidance Paradigm. Thalamic neuronal activity was modulated after injecting an adeno-associated virus (AAV) expressing an engineered acetylcholine Gi-protein coupled receptor. This receptor inhibits neuronal firing when bound by clozapine-n-oxide (CNO). VGAT expression was attenuated in the thalamus by injecting an AAV construct that expressed a VGAT silencing shRNA. VZV induced nociception was significantly decreased after administering CNO in male rats. Nociception significantly increased concomitant with increased thalamic c-fos expression after attenuating thalamic VGAT expression. These data establish that the lateral thalamus (posterior, ventral posteromedial, ventral posterolateral and/or reticular thalamic nucleus) controls VZV induced nociception in the orofacial region, and that GABA in this region appears to reduce the response to VZV induced nociception possibly by gating facial pain input.

Background Most people are initially infected with varicella zoster virus (VZV) at a young age and this infection results in chickenpox. VZV then becomes latent and reactivates later in life resulting in herpes zoster (HZ) or “shingles”. Often VZV infects neurons of the trigeminal ganglia to cause ocular problems, orofacial disease and occasionally a chronic pain condition termed post-herpetic neuralgia (PHN). To date, no model has been developed to study orofacial pain related to varicella zoster. Importantly, the incidence of zoster associated pain and PHN is known to be higher in women, although reasons for this sex difference remain unclear. Prior to this work, no animal model was available to study these sex-differences. Our goal was to develop an orofacial animal model for zoster associated pain which could be utilized to study the mechanisms contributing to this sex difference. Methods To develop this model VZV was injected into the whisker pad of rats resulting in IE62 protein expression in the trigeminal ganglia; IE62 is an immediate early gene in the VZV replication program. Results Similar to PHN patients, rats showed retraction of neurites after VZV infection. Treatment of rats with gabapentin, an agent often used to combat PHN, ameliorated the pain response after whisker pad injection. Aversive behavior was significantly greater for up to 7 weeks in VZV injected rats over control inoculated rats. Sex differences were also seen such that ovariectomized and intact female rats given the lower dose of VZV showed a longer affective response than male rats. The phase of the estrous cycle also affected the aversive response suggesting a role for sex steroids in modulating VZV pain. Conclusions These results suggest that this rat model can be utilized to study the mechanisms of 1) orofacial zoster associated pain and 2) the sex differences underlying zoster associated pain.

SUMMARY The antigen-presenting molecule MR1 presents microbial metabolites related to vitamin B2 biosynthesis to mucosal-associated invariant T cells (MAIT cells). Although bacteria and fungi drive the MR1 biosynthesis pathway, viruses have not previously been implicated in MR1 expression or its antigen presentation. We demonstrate that several herpesviruses inhibit MR1 cell surface upregulation, including a potent inhibition by herpes simplex virus type 1 (HSV-1). This virus profoundly suppresses MR1 cell surface expression and targets the molecule for proteasomal degradation, whereas ligand-induced cell surface expression of MR1 prior to infection enables MR1 to escape HSV-1-dependent targeting. HSV-1 downregulation of MR1 is dependent on de novo viral gene expression, and we identify the Us3 viral gene product as functioning to target MR1. Furthermore, HSV-1 downregulation of MR1 disrupts MAIT T cell receptor (TCR) activation. Accordingly, virus-mediated targeting of MR1 defines an immunomodulatory strategy that functionally disrupts the MR1-MAIT TCR axis., Graphical Abstract, In Brief The antigen-presenting molecule MR1 presents bacterial and fungal metabolites to MAIT cells. McSharry et al. show that the herpesviruses HSV-1 and CMV disrupt MR1 expression. Downregulation of MR1 by HSV-1 inhibits bacterially driven MAIT TCR-dependent activation. This provides evidence of virus immunomodulatory control of the MR1-restricted immune response.

Varicella zoster virus (VZV) infects the face and can result in chronic, debilitating pain. The mechanism for this pain is unknown and current treatment is often not effective, thus investigations into the pain pathway become vital. Pain itself is multidimensional, consisting of sensory and affective experiences. One of the primary brain substrates for transmitting sensory signals in the face is the ventral posterior medial/posterior lateral thalamus (VPM/VPL). In addition, the anterior cingulate cortex (ACC) has been shown to be vital in the affective experience of pain, so investigating both of these areas in freely behaving animals was completed to address the role of the brain in VZV-induced pain. Our lab has developed a place escape avoidance paradigm (PEAP) to measure VZV-induced affective pain in the orofacial region of the rat. Using this assay as a measure of the affective pain experience a significant response was observed after VZV injection into the whisker pad and after VZV infusion into the trigeminal ganglion. Local field potentials (LFPs) are the summed electrical current from a group of neurons. LFP in both the VPM/VPL and ACC was attenuated in VZV injected rats after inhibition of neuronal activity. This inhibition of VPM/VPL neurons was accomplished using a designer receptor exclusively activated by a designer drug (DREADD). Immunostaining showed that cells within the VPM/VPL expressed thalamic glutamatergic vesicle transporter-2, NeuN and DREADD suggesting inhibition occurred primarily in excitable neurons. From these results we conclude: (1) that VZV associated pain does not involve a mechanism exclusive to the peripheral nerve terminals, and (2) can be controlled, in part, by excitatory neurons within the VPM/VPL that potentially modulate the affective experience by altering activity in the ACC.

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.

Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8 + T cells specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8 + T cells are lost over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter (gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was significantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB- and rescuant-infected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures, suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.

We show here that the varicella-zoster virus (VZV) open reading frame 66 (ORF66) protein kinase is one mechanism employed to reduce class I major histocompatibility complex (MHC-I) surface expression in VZV-infected cells. Cells expressing enhanced green fluorescent protein-tagged functional and inactivated ORF66 (GFP-66 and GFP-66kd) from replication-defective adenovirus vectors revealed that ORF66 reduced MHC-I surface levels in a manner dependent on kinase activity. Cells infected with recombinant VZV expressing GFP-66 exhibited a significantly greater reduction in MHC-I surface expression than that observed in cells infected with VZV disrupted in GFP-66 expression. MHC-I maturation was delayed in its transport from the endoplasmic reticulum through the Golgi in both adenovirus-transduced cells expressing only GFP-66 and in VZV-infected cells expressing high levels of GFP-66, and this was predominantly kinase dependent. MHC-I levels were reduced in VZV-infected cells, and analyses of intracellular MHC-I revealed accumulation of folded MHC-I in the Golgi region, irrespective of ORF66 expression. Thus, the ORF66 kinase is important for VZV-mediated MHC-I downregulation, but additional mechanisms also may be involved. Analyses of the VZV ORF9a protein, the ortholog of the bovine herpesvirus 1 transporter associated with antigen processing inhibitor UL49.5 revealed no effects on MHC-I. These results establish a new role for viral protein kinases in immune evasion and suggest that VZV utilizes unique mechanisms to inhibit antigen presentation.

The Amaryllidaceae alkaloid trans-dihydrolycoricidine 7 and three analogues 8-10 were produced via asymmetric chemical synthesis. Alkaloid 7 proved superior to acyclovir, the current standard for herpes simplex virus, type 1 (HSV-1) infection. Compound 7 potently inhibited lytic HSV-1 infection, significantly reduced HSV-1 reactivation, and more potently inhibited varicella zoster virus (VZV) lytic infection. A configurationally defined (3R)-secondary alcohol at C3 proved crucial for efficacious inhibition of lytic HSV-1 infection.

Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of 'quiescent' HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo.

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.

A significant fraction of patients with herpes zoster, caused by Varicella Zoster Virus (VZV), experience chronic pain termed postherpetic neuralgia (PHN). VZV-inoculated rats develop prolonged nocifensive behaviors and serve as a model of PHN. We demonstrate that primary rat cultures show a post-entry block for VZV replication, suggesting the rat is not fully permissive. However, footpads of VZV infected animals show reduced peripheral innervation and innervating dorsal root ganglia (DRG) contained VZV DNA and transcripts of candidate immediate early and early genes. The VZV-infected DRG showed changes in host gene expression patterns, with 84 up-regulated and 116 down-regulated genes seen in gene array studies. qRT-PCR validated the modulation of nociception-associated genes Ntrk2, Trpv1, and Calca (CGRP). The data suggests that VZV inoculation of the rat results in a single round, incomplete infection that is sufficient to induce pain behaviors, and this involves infection of and changes induced in neuronal populations.

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. IMPORTANCE Superinfection 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.

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 protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro , suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.

Reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8 + T cells can completely inhibit HSV reactivation in mice, with interferon-γ affording a portion of this protection. We found that CD8 + T cell lytic granules are also required for the maintenance of neuronal latency both in vivo and in ex vivo ganglia cultures and that their directed release to the junction with neurons in latently infected ganglia did not induce neuronal apoptosis. Here, we describe a nonlethal mechanism of viral inactivation in which the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression.