RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Córnea/inervação , Córnea/metabolismo , Ceratite Herpética/etiologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fibras Adrenérgicas , Animais , Córnea/imunologia , Córnea/virologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Herpesvirus Humano 1 , Humanos , Imunofenotipagem , Ceratite Herpética/metabolismo , Ceratite Herpética/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Depleção Linfocítica , Camundongos , Neurite (Inflamação) , Índice de Gravidade de DoençaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Neuralgia Pós-Herpética/virologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Replicação Viral/fisiologia , Animais , Herpesvirus Humano 3 , Masculino , Neurônios/virologia , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Dor Facial , Herpes Zoster , Herpesvirus Humano 3 , Fatores Sexuais , Animais , Modelos Animais de Doenças , Feminino , Masculino , RatosRESUMO
Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.
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Diferenciação Celular , Varicela/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 3/fisiologia , Queratinócitos/metabolismo , RNA Viral/biossíntese , Proteínas Virais/biossíntese , Replicação Viral/fisiologia , Varicela/genética , Feminino , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , Masculino , RNA Viral/genética , Análise de Sequência de RNA , Proteínas Virais/genéticaRESUMO
UNLABELLED: 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.
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Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 3/fisiologia , Neurônios/virologia , Interferência Viral , Células Cultivadas , Fibroblastos/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 3/crescimento & desenvolvimento , HumanosRESUMO
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.
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Células-Tronco Embrionárias/virologia , Herpesvirus Humano 3/fisiologia , Neurônios/virologia , Células-Tronco Pluripotentes/virologia , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Herpesvirus Humano 3/genética , Humanos , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Replicação ViralRESUMO
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.
Assuntos
Alphaherpesvirinae/enzimologia , Regulação da Expressão Gênica , Proteínas Associadas à Matriz Nuclear/metabolismo , Fases de Leitura Aberta/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Alphaherpesvirinae/classificação , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Linhagem Celular , Fibroblastos/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Suídeo 1/enzimologia , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Herpesvirus Humano 3/enzimologia , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/metabolismo , Humanos , Rim/citologia , Rim/virologia , Proteínas Associadas à Matriz Nuclear/genética , Fosforilação , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genéticaRESUMO
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.
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Axônios/virologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Herpesvirus Humano 3/patogenicidade , Neurônios/virologia , Transporte Axonal , Axônios/ultraestrutura , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiologia , Humanos , Microscopia Eletrônica de Transmissão , Neurônios/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Liberação de VírusRESUMO
Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.
Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Herpesvirus Humano 3/fisiologia , Neurônios/virologia , Fases de Leitura Aberta/genética , Antivirais/farmacologia , Linhagem Celular , Descoberta de Drogas , Herpesvirus Humano 3/efeitos dos fármacos , Células-Tronco Embrionárias Humanas , Humanos , Proteínas Imediatamente Precoces , Transativadores , Proteínas do Envelope Viral , Latência Viral , Replicação ViralRESUMO
There is a continued need to understand varicella-zoster virus (VZV) pathogenesis and to develop more effective antivirals, as it causes chickenpox and zoster. As a human-restricted alphaherpesvirus, the use of human skin in culture and mice is critical in order to reveal the important VZV genes that are required for pathogenesis but that are not necessarily observed in the cell culture. We previously used VZV-expressing firefly luciferase (fLuc), under the control of the constitutively active SV40 promoter (VZV-BAC-Luc), to measure the VZV spread in the same sample. However, the fLuc expression was independent of viral gene expression and viral DNA replication programs. Here, we developed robust reporter VZV viruses by using bacterial artificial chromosome (BAC) technology, expressing luciferase from VZV-specific promoters. We also identified two spurious mutations in VZV-BAC that were corrected for maximum pathogenesis. VZV with fLuc driven by ORF57 showed superior growth in cells, human skin explants, and skin xenografts in mice. The ORF57-driven luciferase activity had a short half-life in the presence of foscarnet. This background was then used to investigate the roles for ORF36 (thymidine kinase (TK)) and ORF13 (thymidylate synthase (TS)) in skin. The studies reveal that VZV-∆TS had increased sensitivity to brivudine and was highly impaired for skin replication. This is the first report of a phenotype that is associated with the loss of TS.
Assuntos
Antivirais , Herpesvirus Humano 3 , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Varicela , Replicação do DNA , DNA Viral , Genes Reporter , Herpes Zoster/patologia , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiologia , Humanos , Luciferases/genética , Camundongos , Camundongos SCID , Pele/patologia , Proteínas Virais Reguladoras e Acessórias/genética , Replicação Viral/genéticaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação Viral da Expressão Gênica , Herpes Simples/imunologia , Herpesvirus Humano 1/patogenicidade , Gânglio Trigeminal/imunologia , Proteínas do Envelope Viral/genética , Animais , Linfócitos T CD8-Positivos/virologia , Chlorocebus aethiops , Feminino , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gânglio Trigeminal/virologia , Células Vero , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
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.
Assuntos
Apresentação de Antígeno/imunologia , Citomegalovirus/fisiologia , Herpesvirus Humano 1/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Ligantes , Masculino , Células T Invariantes Associadas à Mucosa/imunologia , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Fibroblastos/virologia , Herpesvirus Humano 3/enzimologia , Herpesvirus Humano 3/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Substituição de Aminoácidos/genética , Núcleo Celular/química , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Herpesvirus Humano 3/crescimento & desenvolvimento , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/genética , Deleção de Sequência , Coloração e Rotulagem , Transativadores/genética , Transativadores/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/genética , Vírion/químicaRESUMO
Herpes zoster or shingles is the result of varicella zoster virus (VZV) infection and often results in chronic pain that lasts for months after visible symptoms subside. Testosterone often attenuates pain in males. Previous work demonstrates ovarian estrogen effects γ-aminobutyric acid (GABA) signaling in the thalamus, reducing pain but the role of testosterone within the thalamus is currently unknown. Because aromatase affects pain and is present in the thalamus we tested a hypothesis that testosterone converted to estrogen in the thalamus attenuates herpes zoster induced pain. To address this hypothesis, male Sprague-Dawley rats received whisker pad injection of either MeWo cells or MeWo cells containing VZV. To reduce aromatase derived estrogen in these animals we injected aromatase inhibitor letrozole systemically or infused it into the thalamus. To test if estrogen was working through the estrogen receptor (ER) agonist, 4, 4', 4â³-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was infused concomitant with letrozole. Motivational and affective pain was measured after letrozole and/or PPT treatment. Vesicular GABA transporter (VGAT) is important in pain signaling. Because estrogen effects VGAT expression we measured its transcript and protein levels after letrozole treatment. Virus injection and letrozole significantly increased the pain response but thalamic infusion of PPT reduced zoster pain. Letrozole increased the number of thalamic neurons staining for phosphorylated ERK (pERK) but decreased VGAT expression. The results suggest in male rats aromatase derived estradiol interacts with the ER to increase VGAT expression and increase neuronal inhibition in the thalamus to attenuate VZV induced pain.
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[This corrects the article DOI: 10.3389/fnint.2018.00046.].
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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 (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.
Assuntos
Herpes Zoster/virologia , Herpesvirus Humano 3 , Neuralgia Pós-Herpética/virologia , Neuralgia/virologia , Animais , Modelos Animais de Doenças , Injeções/métodos , Masculino , Ratos Sprague-DawleyRESUMO
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.
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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.
RESUMO
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.
Assuntos
Herpesvirus Humano 3/fisiologia , Neuralgia Pós-Herpética/patologia , Neurônios/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Gânglios Espinais/patologia , Gânglios Espinais/virologia , Perfilação da Expressão Gênica , Masculino , Análise em Microsséries , Dados de Sequência Molecular , Neuralgia Pós-Herpética/virologia , Neurônios/virologia , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
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.