RESUMEN
BACKGROUND: The hexapeptide SLIGRL-amide activates protease-activated receptor-2 (PAR-2) and mas-related G protein-coupled receptor C11 (MRGPRC11), both of which are known to be expressed on populations of sensory nerves. SLIGRL-amide has recently been reported to inhibit influenza A (IAV) infection in mice independently of PAR-2 activation, however the explicit roles of MRGPRC11 and sensory nerves in this process are unknown. Thus, the principal aim of this study was to determine whether SLIGRL-amide-induced inhibition of influenza infection is mediated by MRGPRC11 and/or by capsaicin-sensitive sensory nerves. METHODS: The inhibitory effect of SLIGRL-amide on IAV infection observed in control mice in vivo was compared to effects produced in mice that did not express MRGPRC11 (mrgpr-cluster∆ (-/-) mice) or had impaired sensory nerve function (induced by chronic pre-treatment with capsaicin). Complementary mechanistic studies using both in vivo and ex vivo approaches investigated whether the anti-IAV activity of SLIGRL-amide was (1) mimicked by either activators of MRGPRC11 (BAM8-22) or by activators (acute capsaicin) or selected mediators (substance P, CGRP) of sensory nerve function, or (2) suppressed by inhibitors of sensory nerve function (e.g. NK1 receptor antagonists). RESULTS: SLIGRL-amide and BAM8-22 dose-dependently inhibited IAV infection in mrgpr-cluster∆ (-/-) mice that do not express MRGPRC11. In addition, SLIGRL-amide and BAM8-22 each inhibited IAV infection in capsaicin-pre-treated mice that lack functional sensory nerves. Furthermore, the anti-IAV activity of SLIGRL-amide was not mimicked by the sensory neuropeptides substance P or CGRP, nor blocked by either NK1 (L-703,606, RP67580) and CGRP receptor (CGRP8-37) antagonists. Direct stimulation of airway sensory nerves through acute exposure to the TRPV1 activator capsaicin also failed to mimic SLIGRL-amide-induced inhibition of IAV infectivity. The anti-IAV activity of SLIGRL-amide was mimicked by the purinoceptor agonist ATP, a direct activator of mucus secretion from airway epithelial cells. Additionally, both SLIGRL-amide and ATP stimulated mucus secretion and inhibited IAV infectivity in mouse isolated tracheal segments. CONCLUSIONS: SLIGRL-amide inhibits IAV infection independently of MRGPRC11 and independently of capsaicin-sensitive, neuropeptide-releasing sensory nerves, and its secretory action on epithelial cells warrants further investigation.
Asunto(s)
Antivirales/farmacología , Capsaicina/farmacología , Virus de la Influenza A/patogenicidad , Neuronas Aferentes/efectos de los fármacos , Oligopéptidos/farmacología , Infecciones por Orthomyxoviridae/prevención & control , Receptores Acoplados a Proteínas G/agonistas , Tráquea/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Predisposición Genética a la Enfermedad , Humanos , Técnicas In Vitro , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Neuronas Aferentes/metabolismo , Neuronas Aferentes/virología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/fisiopatología , Infecciones por Orthomyxoviridae/virología , Fragmentos de Péptidos/farmacología , Fenotipo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Tráquea/inervación , Tráquea/metabolismo , Tráquea/virologíaRESUMEN
Primary pain and touch sensory neurons not only detect internal and external sensory stimuli, but also receive inputs from other neurons. However, the neuronal derived inputs for primary neurons have not been systematically identified. Using a monosynaptic rabies viruses-based transneuronal tracing method combined with sensory-specific Cre-drivers, we found that sensory neurons receive intraganglion, intraspinal, and supraspinal inputs, the latter of which are mainly derived from the rostroventral medulla (RVM). The viral-traced central neurons were largely inhibitory but also consisted of some glutamatergic neurons in the spinal cord and serotonergic neurons in the RVM. The majority of RVM-derived descending inputs were dual GABAergic and enkephalinergic (opioidergic). These inputs projected through the dorsolateral funiculus and primarily innervated layers I, II, and V of the dorsal horn, where pain-sensory afferents terminate. Silencing or activation of the dual GABA/enkephalinergic RVM neurons in adult animals substantially increased or decreased behavioral sensitivity, respectively, to heat and mechanical stimuli. These results are consistent with the fact that both GABA and enkephalin can exert presynaptic inhibition of the sensory afferents. Taken together, this work provides a systematic view of and a set of tools for examining peri- and extrasynaptic regulations of pain-afferent transmission.
Asunto(s)
Vías Aferentes/fisiología , Vías Eferentes/fisiología , Red Nerviosa/fisiología , Nocicepción/fisiología , Células Receptoras Sensoriales/fisiología , Asta Dorsal de la Médula Espinal/citología , Animales , Virus Defectuosos/fisiología , Encefalinas/fisiología , Miembro Anterior/inervación , Neuronas GABAérgicas/fisiología , Neuronas GABAérgicas/virología , Ganglios Espinales/citología , Hiperalgesia/fisiopatología , Interneuronas/fisiología , Interneuronas/virología , Proteínas del Tejido Nervioso/análisis , Conducción Nerviosa , Neuronas Aferentes/fisiología , Neuronas Aferentes/virología , Neuronas Eferentes/fisiología , Neuronas Eferentes/virología , Nociceptores/fisiología , Células del Asta Posterior/fisiología , Células del Asta Posterior/virología , Terminales Presinápticos/fisiología , Virus de la Rabia/fisiología , Células Receptoras Sensoriales/clasificación , Células Receptoras Sensoriales/virología , Piel/inervación , Asta Dorsal de la Médula Espinal/fisiología , Asta Dorsal de la Médula Espinal/ultraestructura , Replicación Viral , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Primary sensory neurons convey information from the external world to relay circuits within the CNS, but the identity and organization of the neurons that process incoming sensory information remains sketchy. Within the CNS, viral tracing techniques that rely on retrograde transsynaptic transfer provide a powerful tool for delineating circuit organization. Viral tracing of the circuits engaged by primary sensory neurons has, however, been hampered by the absence of a genetically tractable anterograde transfer system. In this study, we demonstrate that rabies virus can infect sensory neurons in the somatosensory system, is subject to anterograde transsynaptic transfer from primary sensory to spinal target neurons, and can delineate output connectivity with third-order neurons. Anterograde transsynaptic transfer is a feature shared by other classes of primary sensory neurons, permitting the identification and potentially the manipulation of neural circuits processing sensory feedback within the mammalian CNS.
Asunto(s)
Transporte Axonal/fisiología , Vías Nerviosas/virología , Neuronas Aferentes/virología , Virus de la Rabia/aislamiento & purificación , Sinapsis/virología , Animales , Ratones , Ratones Transgénicos , Vías Nerviosas/metabolismo , Sinapsis/metabolismoRESUMEN
In contrast to the uterus, the cervix is well innervated during pregnancy and the density of nerve fibers increases before birth. To assess neural connections between the cervix and the spinal cord, the cervix of pregnant mice was injected with the trans-synaptic retrograde neural tract tracer pseudorabies virus (PRV). After 5 days, the virus was present in nerve cells and fibers in specific areas of the sensory, autonomic, and motor subdivisions of the thoracolumbar spinal cord. In nonpregnant controls, the virus was predominantly distributed in laminae I-III in the dorsal gray sensory areas with the heaviest label in the substantia gelatinosa compared with the autonomic or motor areas. Labeled cells and processes were sparse in other regions, except for a prominent cluster in the intermediolateral column (lamina VII). Photomicrographs of spinal cord sections were digitized, and the total area with the virus was estimated. Compared with nonpregnant controls, the area with PRV was significantly decreased in all the spinal cord subdivisions in pregnant mice except in the intermediolateral column. However, areas with the virus were equivalent in mice injected with PRV at 4 days or 1 day before birth. These findings suggest that the predominant innervation of the murine cervix is from the sensory regions of the thoracolumbar spinal cord, and that these connections diminish with pregnancy. The results raise the possibility that the remaining connections from sensory and autonomic subdivisions, particularly the intermediolateral column, of the thoracolumbar spinal cord may be important for increased density of nerve fibers in the cervix as pregnancy nears term.
Asunto(s)
Cuello del Útero/inervación , Vías Nerviosas/fisiología , Técnicas de Trazados de Vías Neuroanatómicas , Preñez , Médula Espinal/fisiología , Animales , Recuento de Células , Cuello del Útero/citología , Cuello del Útero/virología , Femenino , Herpesvirus Suido 1/fisiología , Ratones , Ratones Endogámicos C3H , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neuronas Motoras/virología , Fibras Nerviosas/fisiología , Fibras Nerviosas/virología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Trazadores del Tracto Neuronal/farmacología , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Neuronas Aferentes/virología , Embarazo , Transmisión Sináptica/fisiologíaRESUMEN
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-gamma 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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Gránulos Citoplasmáticos/enzimología , Granzimas/metabolismo , Herpesvirus Humano 1/fisiología , Queratitis Herpética/virología , Neuronas Aferentes/virología , Latencia del Virus , Animales , Apoptosis , Células Cultivadas , Gránulos Citoplasmáticos/inmunología , Herpesvirus Humano 1/inmunología , Proteínas Inmediatas-Precoces/metabolismo , Queratitis Herpética/inmunología , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/citología , Técnicas de Cultivo de Tejidos , Ganglio del Trigémino/citología , Ganglio del Trigémino/virología , Activación ViralRESUMEN
Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Herpesvirus Humano 2/genética , MicroARNs/metabolismo , Neuronas Aferentes/virología , Proteínas Virales/metabolismo , Latencia del Virus/genética , Adulto , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Cartilla de ADN/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/virología , Componentes del Gen , Cobayas , Humanos , Luciferasas , Masculino , MicroARNs/genética , Microscopía Fluorescente , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The pathways involved in the emotional aspects of thirst, the arousal and affect associated with the generation of thirst and the motivation to obtain satiation, have been studied but remain poorly understood. Rats were therefore injected with the neurotropic virus pseudorabies in either the insular or cingulate cortex. After 2 days of infection, pseudorabies-positive neurons were identified within the thalamus and lamina terminalis. In a separate group of rats, the retrograde tracer cholera toxin subunit b (CTb) was used in combination with either isotonic (0.15 M NaCl) or hypertonic (0.8 M NaCl) saline (1 ml/100 g body wt ip). Rats injected with CTb in the insular cortex and stimulated with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the paraventricular, rhomboid, and reuniens thalamic nuclei, whereas those rats injected with CTb in the cingulate cortex and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the medial part of the mediodorsal, interanteromedial, anteromedial, and ventrolateral part of the laterodorsal thalamic nuclei. Rats injected with CTb in the dorsal midline of the thalamus and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons within the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and insular cortex but not the subfornical organ. A small proportion of the CTb-positive neurons in the OVLT were immunopositive for transient receptor potential vanilloid 1, a putative osmoresponsive membrane protein. These results identify functional thalamocortical pathways involved in relaying osmotic signals to the insular and cingulate cortex and may provide a neuroanatomical framework for the emotional aspects of thirst.
Asunto(s)
Corteza Cerebral/metabolismo , Hipotálamo/metabolismo , Neuronas Aferentes/metabolismo , Núcleos Talámicos/metabolismo , Sed , Equilibrio Hidroelectrolítico , Animales , Corteza Cerebral/citología , Corteza Cerebral/virología , Toxina del Cólera/metabolismo , Herpesvirus Suido 1/aislamiento & purificación , Hipotálamo/citología , Hipotálamo/virología , Soluciones Isotónicas , Masculino , Vías Nerviosas/metabolismo , Neuronas Aferentes/virología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Solución Salina Hipertónica/metabolismo , Transducción de Señal , Cloruro de Sodio/metabolismo , Coloración y Etiquetado/métodos , Canales Catiónicos TRPV/metabolismo , Núcleos Talámicos/citología , Núcleos Talámicos/virologíaRESUMEN
The latent persistence of herpes simplex virus type 1 (HSV-1) in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltrate. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to characterize the immune cell infiltrates by this feature. We combined in situ hybridization, laser cutting microscopy, and single cell RT-PCR to demonstrate the expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T-cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8+ T-cells had features of the memory effector phenotype. The voltage-gated potassium channel Kv1.3, a marker of chronic activated memory effector cells, and the chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells. The corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all CD8 T-cells. In addition, T-cells expressed granzymes and perforin. In contrast to animal models of HSV-1 latency, hardly any FoxP3-positive regulatory T-cells were detected in human TG. Thus, HSV-1 IE genes are expressed in human TG and the infiltrating T-cells bear several characteristics that suggest viral antigenic stimulation.
Asunto(s)
Genes Inmediatos-Precoces/genética , Herpes Simple/genética , Herpes Simple/virología , Herpesvirus Humano 1/genética , Linfocitos T/virología , Ganglio del Trigémino/virología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Quimiocinas/inmunología , Quimiocinas/metabolismo , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Células Clonales/inmunología , Células Clonales/virología , Femenino , Regulación Viral de la Expresión Génica/genética , Genes Virales/genética , Herpes Simple/fisiopatología , Herpesvirus Humano 1/inmunología , Humanos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Canal de Potasio Kv1.3/metabolismo , Masculino , Persona de Mediana Edad , Neuronas Aferentes/inmunología , Neuronas Aferentes/virología , Fenotipo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Linfocitos T/inmunología , Ganglio del Trigémino/citología , Ganglio del Trigémino/inmunología , Latencia del Virus/genética , Latencia del Virus/inmunologíaRESUMEN
Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.
Asunto(s)
Ganglios Espinales/virología , Neuronas Aferentes/virología , Virus de la Rabia/ultraestructura , Rabia/virología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Neuronas Aferentes/ultraestructura , Virus de la Rabia/fisiología , Factores de Tiempo , Ensamble de VirusRESUMEN
The reeler mouse is an autosomal recessive mutant mouse caused by mutation of the reelin gene and characterized by cerebellar ataxia. To determine whether the distribution pattern of precerebellar nuclei neurons in the brainstem of the reeler mouse changes, we injected a small volume of a replication-defective recombinant adenovirus carrying E. coli beta-galactosidase (lacZ) into the cerebellar cortex of normal and reeler mice. Five days later, the mice were transcardially perfused by a fixative solution. X-gal staining of coronal or sagittal sections of the brainstem revealed that many origins for reticulocerebellar, cuneocerebellar, trigeminocerebellar, and pontocerebellar projections were retrogradely labeled, but only a few olivocerebellar neurons were labeled. Retrogradely labeled neurons in the lateral reticular nucleus tended to locate more laterally and be more condensed into a small compartment in the reeler compared with their normal counterparts. Retrogradely labeled neurons in the external cuneate nucleus were more dorsally shifted in the reeler mice compared with their normal counterparts. We could not find any differences between the normal and reeler mice in the distribution patterns of their trigeminocerebellar projection neurons. Retrogradely labeled pontocerebellar neurons in the basilar pons of the reeler mouse were reduced in number compared with their normal counterparts in addition to being more ventrally and laterally shifted. These findings strongly suggest that the migration of some precerebellar nuclei neurons from the rhombic lip to their final loci may be obstructed in the reeler mice.
Asunto(s)
Adenoviridae , Núcleo Celular/virología , Corteza Cerebelosa/citología , Ratones Mutantes Neurológicos/genética , Neuronas Aferentes/citología , Adenoviridae/genética , Infecciones por Adenoviridae/genética , Animales , Corteza Cerebelosa/virología , Femenino , Operón Lac , Masculino , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Neuronas Aferentes/metabolismo , Neuronas Aferentes/virología , Núcleo Olivar/citología , Núcleo Olivar/metabolismo , Núcleo Olivar/virología , Proteína Reelina , Coloración y Etiquetado , beta-Galactosidasa/metabolismoRESUMEN
Primary cultures were made from adult mouse spinal ganglia for depicting an ultrastructural description of rabies virus (RABV) infection in adult mouse sensory neuron cultures; they were infected with rabies virus for 24, 36, and 48 h. The monolayers were processed for transmission electron microscopy and immunochemistry studies at the end of each period. As previously reported, sensory neurons showed great susceptibility to infection by RABV; however, in none of the periods evaluated were assembled virions observed in the cytoplasm or seen to be associated with the cytoplasmic membrane. Instead, fibril matrices of aggregated ribonucleoprotein were detected in the cytoplasm. When infected culture lysate were inoculated into normal animals via intra-cerebral route it was observed that these animals developed clinical symptoms characteristic of infection and transmission electron microscopy revealed assembled virions in the cerebral cortex and other areas of the brain. Sensory neurons infected in vitro by RABV produced a large amount of unassembled viral ribonucleoprotein. However, this intracellular material was able to produce infection and virions on being intra-cerebrally inoculated. It can thus be suggested that the lack of intracellular assembly in sensory neurons forms part of an efficient dissemination strategy.
Asunto(s)
Animales , Ratones , Ganglios Espinales/virología , Neuronas Aferentes/virología , Virus de la Rabia/ultraestructura , Rabia/virología , Células Cultivadas , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/ultraestructura , Microscopía Electrónica de Transmisión , Neuronas Aferentes , Virus de la Rabia/fisiología , Factores de Tiempo , Ensamble de VirusRESUMEN
This work was aimed at the morphological and biochemical characterisation of the most susceptible neuronal subpopulation to rabies virus (RABV) infection. Adult mouse DRG cultures were infected with RABV and double-processed for viral antigen detection and neuropeptides: calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). It was found that 56% of the neurons in culture were small (diameter < 20 microm) but, in spite of this, 69% of the infected neurons had intermediate and large diameters (> or = 20 microm). More than 50% of infected neurons expressed NPY, VIP or SP, whereas no association was found between infected neurons and the presence of CGRP or GAL. Despite SP having been shown to be a small neuron marker, it was found that RABV infects medium and large-sized SP positive cells. RABV preference for larger neurons could explain part of the neuropathogenesis since it can be suggested that, following a rabid accident, the virus uses large neurons (mainly innervating muscle and joints) in vivo to be transported later on to the central nervous system.
Asunto(s)
Neuronas Aferentes/metabolismo , Neuronas Aferentes/patología , Neuronas Aferentes/virología , Rabia/patología , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Galanina/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/virología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos ICR , Neuropéptido Y/metabolismo , Rabia/metabolismo , Virus de la Rabia , Sustancia P/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.
Asunto(s)
Farmacorresistencia Viral/genética , Ganglios Espinales/virología , Herpesvirus Humano 1/genética , Mutación Puntual , Timidina Quinasa/genética , Núcleos del Trigémino/virología , Proteínas Virales/genética , Aciclovir/uso terapéutico , Antivirales/uso terapéutico , Ganglios Espinales/enzimología , Ganglios Espinales/patología , Herpes Simple/enzimología , Herpes Simple/genética , Herpes Simple/patología , Herpesvirus Humano 1/enzimología , Humanos , Neuronas Aferentes/enzimología , Neuronas Aferentes/patología , Neuronas Aferentes/virología , Timidina Quinasa/metabolismo , Núcleos del Trigémino/enzimología , Núcleos del Trigémino/patología , Proteínas Virales/metabolismo , Latencia del Virus/efectos de los fármacos , Latencia del Virus/genéticaRESUMEN
Sensory neurons of the trigeminal ganglion (TG) are of crucial importance in the pathogenesis of many alphaherpesviruses, constituting major target cells for latency and reactivation events. We showed earlier that a subpopulation of porcine TG neurons, in contrast to other porcine cell types, is highly resistant to cell death induced by infection with the porcine alphaherpesvirus pseudorabies virus (PRV). Here, we report that expression of Brn-3a, a neuron-specific transcription factor implicated in cell survival of sensory neurons, correlates with the increased resistance of TG neurons towards PRV-induced cell death. In addition, overexpression of Brn-3a in the sensory neuronal cell line ND7 markedly increased resistance of these cells to PRV-induced cell death. Hence, Brn-3a may play a hitherto uncharacterized role in protection of sensory neurons from alphaherpesvirus-induced cell death, which may have implications for different aspects of the alphaherpesvirus life cycle, including latency/reactivation events.
Asunto(s)
Herpesvirus Suido 1/patogenicidad , Neuronas Aferentes/virología , Factor de Transcripción Brn-3A/biosíntesis , Ganglio del Trigémino/virología , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , Células Cultivadas , Expresión Génica , Microscopía Confocal , Neuronas Aferentes/citología , Ratas , Porcinos , Factor de Transcripción Brn-3A/fisiología , Ganglio del Trigémino/citologíaRESUMEN
Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons. HSV-1 establishes a life-long latent infection in these neurons, during which the transcription of the viral genome is silent, except for the sequences encoding the latency-associated transcript (LAT). To determine if HSV-1 latency might affect calcitonin gene-related peptide (CGRP) expression in trigeminal sensory neurons, we transfected primary neuronal cultures of trigeminal ganglia from rat embryos with plasmids expressing LAT. In the presence of Bone Morphogenetic Protein-7 (BMP7), CGRP was expressed in 49% of sensory neurons. However, this percentage was reduced to 19% in neurons transfected with LAT expressing plasmids. We also found that transfection of the IE63 gene of varicella-zoster virus (VZV) reduced the percentage of trigeminal neurons containing CGRP. However, the observed effect of IE63 in contrast to that of LAT was completely reversed by treatment of cultures with MgCl2, which indicates that the effect of IE63 was due to increased release of CGRP from trigeminal neurons. We provide here the first evidence that HSV-1 LAT decreases the level of CGRP in trigeminal neurons. These effects may be important for reducing the neuroinflammatory response, thus protecting host neuronal cells during HSV-1 latency in trigeminal neurons. In contrast, increased release of CGRP in the presence of IE63 protein may contribute to the neuralgias associated with VZV infection.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Neuronas Aferentes/virología , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Animales , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/farmacología , Supervivencia Celular/fisiología , Células Cultivadas , ADN Viral/genética , ADN Viral/farmacología , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Ganglios Espinales/virología , Eliminación de Gen , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/fisiología , Herpes Simple/fisiopatología , Proteínas Inmediatas-Precoces/metabolismo , Cloruro de Magnesio/farmacología , MicroARNs , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Transfección , Factor de Crecimiento Transformador beta/farmacología , Ganglio del Trigémino/citología , Proteínas del Envoltorio Viral/metabolismo , Latencia del Virus/fisiologíaRESUMEN
A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.
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Apoptosis , Neuronas Aferentes/citología , Neuronas Aferentes/virología , Neuronas Receptoras Olfatorias/virología , Proteínas Serina-Treonina Quinasas/fisiología , Simplexvirus/enzimología , Proteínas Virales/fisiología , Órgano Vomeronasal/virología , Animales , Peso Corporal , Encéfalo/virología , Modelos Animales de Enfermedad , Encefalitis/virología , Femenino , Eliminación de Gen , Herpes Simple/inmunología , Herpes Simple/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Nervio Olfatorio/virología , Neuronas Receptoras Olfatorias/citología , Proteínas Serina-Treonina Quinasas/genética , Simplexvirus/genética , Simplexvirus/inmunología , Simplexvirus/patogenicidad , Análisis de Supervivencia , Proteínas Virales/genética , Órgano Vomeronasal/citologíaRESUMEN
BACKGROUND: To examine the role of inflammatory mediators in neuropathic pain, we used a replication-defective genomic herpes simplex virus (HSV)-based vector containing the coding sequence for the anti-inflammatory peptide interleukin (IL)-4 under the transcriptional control of the HSV ICP4 immediate early promoter, vector S4IL4, to express IL-4 in dorsal root ganglion (DRG) neurons in vivo. RESULTS: Subcutaneous inoculation of S4IL4 in the foot transduced lumbar DRG to produce IL-4. Transgene-mediated expression of IL-4 did not alter thermal latency or tactile threshold in normal animals, but inoculation of S4IL4 1 week after spinal nerve ligation (SNL) reduced mechanical allodynia and reversed thermal hyperalgesia resulting from SNL. Inoculation of S4IL4 1 week before SNL delayed the development of thermal hyperalgesia and tactile allodynia, but did not prevent the ultimate development of these manifestations of neuropathic pain. S4IL4 inoculation suppressed non-noxious-induced expression of c-Fos immunoreactivity in dorsal horn of spinal cord and reversed the upregulation of spinal IL-1beta, PGE2, and phosphorylated-p38 MAP kinase, characteristic of neuropathic pain. CONCLUSION: HSV-mediated expression of IL-4 effectively reduces the behavioral manifestations of neuropathic pain, and reverses some of the biochemical and histologic correlates of neuropathic pain at the spinal level.
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Ganglios Espinales/inmunología , Interleucina-4/inmunología , Neuralgia/inmunología , Neuronas Aferentes/inmunología , Enfermedades del Sistema Nervioso Periférico/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/virología , Vectores Genéticos/genética , Hiperalgesia/inmunología , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Interleucina-4/genética , Interleucina-4/metabolismo , Masculino , Neuralgia/metabolismo , Neuralgia/fisiopatología , Neuronas Aferentes/metabolismo , Neuronas Aferentes/virología , Umbral del Dolor/fisiología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Células del Asta Posterior/inmunología , Células del Asta Posterior/metabolismo , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/genética , Tiempo de Reacción/inmunología , Simplexvirus/genética , Nervios Espinales/lesiones , Nervios Espinales/fisiopatología , Nervios Espinales/cirugía , Transfección/métodos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We have demonstrated that dorsal root ganglion neurons transduced with a recombinant replication-defective herpes simplex virus vector coding for glutamic acid decarboxylase (QHGAD67) release GABA to produce an analgesic effect in rodent models of pain. In this study, we examined the mechanism of transgene-mediated GABA release from dorsal root ganglion neurons in vitro and in vivo. Release of GABA from dorsal root ganglion neurons transduced with QHGAD67 was not increased by membrane depolarization induced by 60 mM extracellular K+ nor reduced by the removal of Ca2+ from the medium. Release of GABA from transduced dorsal root ganglion neurons was, however, blocked in a dose-dependent manner by NO-711, a selective inhibitor of the GABA transporter-1. The amount of GABA released from a spinal cord slice preparation, prepared from animals transduced by subcutaneous inoculation of QHGAD67 in the hind paws, was substantially increased compared to animals transduced with control vector Q0ZHG or normal animals, but the amount of GABA released was not changed by stimulation of the dorsal roots at either low (0.1 mA, 0.5-ms duration) or high (10 mA, 0.5-ms duration) intensity. We conclude that QHGAD67-mediated GABA release from dorsal root ganglion neurons is non-vesicular, independent of electrical depolarization, and that this efflux is mediated through reversal of the GABA transporter.
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Glutamato Descarboxilasa/metabolismo , Isoenzimas/metabolismo , Neuronas Aferentes/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Ganglios Espinales/citología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Ácido Glutámico/farmacología , Glutamina/análogos & derivados , Glutamina/farmacología , Isoenzimas/genética , Isoenzimas/inmunología , Neuronas Aferentes/virología , Ácidos Nipecóticos/farmacología , Oximas/farmacología , Ratas , Ratas Sprague-Dawley , Simplexvirus/genética , Nervios Espinales/fisiología , Nervios Espinales/efectos de la radiación , Transfección/métodos , Tubulina (Proteína)/metabolismoRESUMEN
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 (rOka deltaORF63) 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.
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Apoptosis , Varicela/virología , Herpesvirus Humano 3/fisiología , Proteínas Inmediatas-Precoces/fisiología , Proteínas del Envoltorio Viral/fisiología , Células Cultivadas , Regulación hacia Abajo , Herpesvirus Humano 3/química , Humanos , Neuronas Aferentes/fisiología , Neuronas Aferentes/virologíaRESUMEN
CD8+ T-cell persistence can be seen in ganglia harboring latent herpes simplex virus (HSV) infection. While there is some evidence that these cells suppress virus reactivation, this view remains controversial. Given that maintenance of latency by CD8+ T cells would necessitate ongoing exposure to antigen within this site, we sought evidence for such chronic stimulation. Initial experiments showed infiltration by activated but not naïve CD8+ T cells into ganglia harboring latent HSV infection. While such infiltration was independent of T-cell specificity, once recruited, only virus-specific T cells expressed high levels of preformed granzyme B, a marker of ongoing activation. Moreover, bone marrow replacement chimeras showed that these elevated granzyme levels were totally dependent on presentation by parenchymal cells within the ganglia. Overall, this study argues that activated CD8+ T cells are nonspecifically recruited into latently infected ganglia, and in this site they are exposed to ongoing antigen stimulation, most likely by infected neuronal cells.