RESUMEN
Rabies is a lethal infectious disease caused by rabies virus (RABV). Fear and anxiety are the distinguished symptoms in rabies patients. Fusion of RABV envelope glycoprotein (RVG) to host cell membrane initiates rabies pathogenesis via interacting with PDZ domain of signaling proteins. We assessed the anxiety-like behaviors, and hypothalamic-pituitary-adrenal axis (HPA) response to RVG infection. Contribution of PDZ binding motif (PBM) of RVG to the observed effects was also examined using a mutant form of RVG, ΔRVG, with deleted last four amino acids at PBM C-terminus. Lentiviral vectors containing RVG and/or ΔRVG genes were injected into the rat brain areas involved in anxiety including hypothalamus, dorsal hippocampus, and amygdala. RVG/ΔRVG neural expression was examined by fluorescent microscopy. Anxiety-like behaviors were assessed by elevated plus maze (EPM) and open field (OF) tasks. HPA response was evaluated via measuring corticosterone serum level by ELISA technique. RVG/ΔRVG were successfully expressed in neurons of the injected areas. RVG, but not ΔRVG, infection of hypothalamus and amygdala increased the time spent in EPM open arms, and OF total distance moved and velocity. RVG, but not ΔRVG, infection of hypothalamus and dorsal hippocampus increased corticosterone level. The anxiety-like behaviors and exploratory/locomotor activities of rats with RVG infection in hypothalamus, and amygdala are mediated by PBM of RVG. The HPA response to RVG infection of hypothalamus and dorsal hippocampus is dependent to PBM of RVG. Triggering anxiety-related signaling by PBM of RVG seems to be one of the mechanisms involved in anxiety behaviors seen in patients with rabies.
Asunto(s)
Virus de la Rabia , Rabia , Animales , Ansiedad , Corticosterona/metabolismo , Glicoproteínas , Humanos , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Virus de la Rabia/genética , Virus de la Rabia/metabolismo , RatasRESUMEN
Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two µl (108 T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat's brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.
Asunto(s)
Virus de la Rabia , Animales , Giro Dentado/metabolismo , Estimulación Eléctrica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/farmacología , Hipocampo/metabolismo , Potenciación a Largo Plazo , Plasticidad Neuronal/fisiología , Virus de la Rabia/metabolismo , RatasRESUMEN
Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.
Asunto(s)
Barrera Hematoencefálica/virología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Modelos Biológicos , Barrera Hematoencefálica/fisiología , Línea Celular , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Encefalitis Japonesa/patología , Encefalitis Japonesa/virología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/virología , Humanos , ARN Viral/genética , ARN Viral/metabolismo , Virulencia , Replicación ViralRESUMEN
Mass vaccination with the live attenuated vaccine YF-17D is the current way to prevent infection with Yellow fever virus (YFV). However, 0.000012-0.00002% of vaccinated patients develop post-vaccination neurological syndrome (YEL-AND). Understanding the factors responsible for neuroinvasion, neurotropism, and neurovirulence of the vaccine is critical for improving its biosafety. The YF-FNV vaccine strain, known to be associated with a higher frequency of YEL-AND (0.3-0.4%) than YF-17D, is an excellent model to study vaccine neuroinvasiveness. We determined that neuroinvasiveness of YF-FNV occured both via infection and passage through human brain endothelial cells. Plaque purification and next generation sequencing (NGS) identified several neuroinvasive variants. Their neuroinvasiveness was not higher than that of YF-FNV. However, rebuilding the YF-FNV population diversity from a set of isolated YF-FNV-N variants restored the original neuroinvasive phenotype of YF-FNV. Therefore, we conclude that viral population diversity is a critical factor for YFV vaccine neuroinvasiveness.
RESUMEN
Rabies is a life-threatening viral infection of the brain. Rabies virus (RABV) merely infects excitable cells including neurons provoking drastic behaviors including negative emotional memories. RABV glycoprotein (RVG) plays a critical role in RABV pathogenesis. RVG interacts with various cytoplasmic PDZ (PSD-95/Dlg/ZO-1) containing proteins through its PDZ binding motif (PBM). PTZ domains have crucial role in formation and function of signal transduction. Hippocampus is one of the cerebral regions that contain high load of viral antigens. We examined impact of RVG expression in the dorsal hippocampus on aversive as well as spatial learning and memory performance in rats. Two microliter of the lentiviral vector (~108 T.U./ml) encoding RVG or ∆RVG (deleted PBM) genomes was microinjected into the hippocampal CA1. After 1 week, rat's brain was cross-sectioned and RVG/∆RVG-expressing neuronal cells were confirmed by fluorescent microscopy. Passive avoidance and spatial learning and memory were assessed in rats by Shuttle box and Morris water maze (MWM). In the shuttle box, both RVG and ∆RVG decreased the time spent in the dark compartment compared to control (p < 0.05). In MWM, RVG and ∆RVG did not affect the acquisition of spatial task. In the probe test, RVG-expressing rats spent more time in the target quadrant, and also reached the platform position sooner than control group (p < 0.05). Rats expressing ∆RVG significantly swam farther from the hidden platform than RVG group (p < 0.05). Our data indicate RVG expression in the hippocampus strengthens aversive and spatial learning and memory performance. The boosting effect on spatial but not avoidance memory is mediated through PBM.
Asunto(s)
Reacción de Prevención , Región CA1 Hipocampal/fisiopatología , Glicoproteínas/genética , Aprendizaje por Laberinto , Virus de la Rabia/genética , Memoria Espacial , Proteínas Virales/genética , Animales , Región CA1 Hipocampal/metabolismo , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicoproteínas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inyecciones Intraventriculares , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Neuronas/metabolismo , Neuronas/patología , Virus de la Rabia/química , Virus de la Rabia/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas Estereotáxicas , Transgenes , Proteínas Virales/metabolismoRESUMEN
Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thr kinase 2 (MAST2) inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2-PDZ domain. Our strategy involved modifying the length and flexibility of the noninteracting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2-PDZ domain, with Kd values decreasing from 1300 to 60 nm, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases.
Asunto(s)
Neuritas/metabolismo , Proyección Neuronal/efectos de los fármacos , Dominios PDZ/fisiología , Estimulantes del Sistema Nervioso Central/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Microtúbulos/metabolismo , Neuronas/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Virus de la Rabia , Relación Estructura-Actividad , Proteínas Virales/metabolismo , Proteínas Virales/farmacologíaRESUMEN
The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Neurológicos , Fármacos Neuroprotectores/metabolismo , Animales , Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Células Cultivadas , Células Endoteliales/fisiología , Humanos , Neuronas/metabolismo , Fármacos Neuroprotectores/administración & dosificaciónRESUMEN
Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies in vitro as a potential therapeutic candidate. The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir (also known as T-705) to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain.
Asunto(s)
Vacunas Antirrábicas/uso terapéutico , Virus de la Rabia/inmunología , Rabia/metabolismo , Anticuerpos de Cadena Única/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Barrera Hematoencefálica/metabolismo , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Rabia/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/inmunología , Virus de la Rabia/patogenicidad , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Live attenuated vaccines have proved to be mostly valuable in the prevention of infectious diseases in humans, especially in developing countries. The safety and potency of vaccine, and the consistency of vaccine batch-to-batch manufacturing, must be proven before being administrated to humans. For now, the tests used to control vaccine safety largely involve animal testing. For live viral vaccines, regulations require suppliers to demonstrate the absence of neurovirulence in animals, principally in non-human primates and mice. In a search to reduce the use of animals and embracing the 3Rs principles (Replacement, Reduction, Refinement in the use of laboratory animals), we developed a new Blood-Brain Barrier Minibrain (BBB-Minibrain) in cellulo device to evaluate the neuroinvasiveness/neurovirulence of live Yellow Fever virus (YFV) vaccines. A pilot study was performed using the features of two distinct YFV strains, with the ultimate goal of proposing a companion test to characterize YFV neurovirulence. Here, we demonstrate that the BBB-Minibrain model is a promising alternative to consider for future replacement of YFV vaccine in vivo neurovirulence testing (see graphical abstract).
Asunto(s)
Barrera Hematoencefálica/metabolismo , Modelos Inmunológicos , Vacuna contra la Fiebre Amarilla , Virus de la Fiebre Amarilla , Barrera Hematoencefálica/virología , Células Cultivadas , Humanos , Proyectos Piloto , Control de Calidad , Vacuna contra la Fiebre Amarilla/inmunología , Vacuna contra la Fiebre Amarilla/farmacocinética , Vacuna contra la Fiebre Amarilla/farmacologíaRESUMEN
In the field of live viral vaccines production, there is an unmet need for in vitro tests complying a 3R approach (Refine, Replace and Reduce the use of animal experimentation) to replace the post-licensing safety tests currently assayed in animals. Here, we performed a pilot study evaluating whether virulence of rabies virus, RABV, can be forecast by an in vitro test of neurite outgrowth. The rationale to use neurite outgrowth as a read-out for this test is based on the salient property of the cytoplasmic domain of the G-protein (Cyto-G) of virulent RABV strains - not of attenuated RABV strains - to stimulate neurite outgrowth in vitro. We observed that neurite elongation triggered by the Cyto-Gs encoded by different RABV field isolates correlate with the distinct virulence scores obtained in a mouse model of experimental rabies. Our results cast the idea that it could be feasible to predict RABV virulence by testing the in vitro property of a RABV strain to promote neurite outgrowth without the use of animal experimentation.
Asunto(s)
Alternativas a las Pruebas en Animales , Glicoproteínas/metabolismo , Neuritas/virología , Fragmentos de Péptidos/metabolismo , Virus de la Rabia , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación Viral de la Expresión Génica/fisiología , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Neuritas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Vacunas Antirrábicas/inmunología , Ratas , Proteínas Recombinantes de Fusión , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Rabies is a fatal zoonotic disease and infections generally lead to a fatal encephalomyelitis in both humans and animals. In South Africa, domestic (dogs) and the wildlife (yellow mongoose) host species maintain the canid and mongoose rabies variants respectively. In this study, pathogenicity differences of South African canid and mongoose rabies viruses were investigated in a murine model, by assessing the progression of clinical signs and survivorship. Comparison of glycoprotein gene sequences revealed amino acid differences that may underpin the observed pathogenicity differences. Cumulatively, our results suggest that the canid rabies virus may be more neurovirulent in mice than the mongoose rabies variant.
Asunto(s)
Virus de la Rabia/genética , Rabia/veterinaria , Animales , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/virología , Perros , Herpestidae , Humanos , Ratones , Filogenia , ARN Viral/genética , Rabia/epidemiología , Rabia/virología , Virus de la Rabia/patogenicidad , Sudáfrica/epidemiología , Virulencia , ZoonosisRESUMEN
The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVGP fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVGP fusion protein was able to cross the in celluloBBB and neutralize RABV.
Asunto(s)
Barrera Hematoencefálica , Glicoproteínas/inmunología , Fragmentos de Péptidos/inmunología , Planticuerpos/farmacología , Virus de la Rabia/inmunología , Proteínas Virales/inmunología , Anticuerpos Neutralizantes/biosíntesis , Línea Celular , Humanos , Planticuerpos/aislamiento & purificación , Planticuerpos/metabolismo , Plantas Modificadas Genéticamente , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión , NicotianaRESUMEN
RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.
Asunto(s)
Antivirales/farmacología , Poliaminas/metabolismo , Virus ARN/efectos de los fármacos , Acetiltransferasas/metabolismo , Animales , Línea Celular , Fiebre Chikungunya/tratamiento farmacológico , Fiebre Chikungunya/virología , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/metabolismo , Brotes de Enfermedades , Ebolavirus/efectos de los fármacos , Ebolavirus/metabolismo , Eflornitina/farmacología , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/virología , Humanos , Ratones , Ratones Endogámicos C57BL , Espermina/análogos & derivados , Espermina/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/virologíaRESUMEN
The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.
Asunto(s)
Proteína Quinasa 12 Activada por Mitógenos/metabolismo , Complejos Multienzimáticos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 4/metabolismo , Transducción de Señal/fisiología , Muerte Celular , Línea Celular Tumoral , Humanos , Proteína Quinasa 12 Activada por Mitógenos/genética , Complejos Multienzimáticos/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 4/genéticaRESUMEN
PDZ (PSD-95/Dlg/ZO-1) domains play a major role in neuronal homeostasis in which they act as scaffold domains regulating cellular trafficking, self-association and catalytic activity of essential proteins such as kinases and phosphatases. Because of their central role in cell signaling, cellular PDZ-containing proteins are preferential targets of viruses to hijack cellular function to their advantage. Here, we describe how the viral G protein of the rabies virus specifically targets the PDZ domain of neuronal enzymes during viral infection. By disrupting the complexes formed by cellular enzymes and their ligands, the virus triggers drastic effect on cell signaling and commitment of the cell to either survival (virulent strains) or death (vaccinal strains). We provide structural and biological evidences that the viral proteins act as competitors endowed with specificity and affinity in an essential cellular process by mimicking PDZ binding motif of cellular partners. Disruption of critical endogenous protein-protein interactions by viral protein drastically alters intracellular protein trafficking and catalytic activity of cellular proteins that control cell homeostasis. This work opens up many perspectives to mimic viral sequences and developing innovative therapies to manipulate cellular homeostasis.
Asunto(s)
Neuronas/metabolismo , Dominios PDZ , Virus de la Rabia/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/enzimología , Neuronas/virología , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 4/metabolismo , Virus de la Rabia/metabolismoRESUMEN
Influenza A virus triggers a contagious respiratory disease that can cause considerable morbidity and mortality. Using an in vitro approach, we previously demonstrated that the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) plays a key role in influenza A virus-mediated immune response. However, the importance of RIG-I signaling in vivo has not been thoroughly examined, because of the lack of an appropriate mouse models. To circumvent this issue, we generated a new transgenic mouse overexpressing LGP2 (hereafter, "LGP2 TG mice"), a major regulator of the RIG-I signaling pathway. The time course of several parameters was compared in infected wild-type and LGP2 TG mice. We found that LGP2 TG mice displayed significantly reduced inflammatory mediators and a lower leukocyte infiltration into the bronchoalveolar airspace. More importantly, LGP2 TG mice had a significant survival advantage. Hence, our in vivo study reveals that LGP2 is a major downregulator of the influenza A virus-triggered detrimental inflammatory response.
Asunto(s)
Interacciones Huésped-Patógeno , Virus de la Influenza A/fisiología , ARN Helicasas/metabolismo , Animales , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Mediadores de Inflamación/análisis , Leucocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Transducción de Señal , Análisis de SupervivenciaRESUMEN
PTEN phosphatase is a tumor suppressor controlling notably cell growth, proliferation and survival. The multisite phosphorylation of the PTEN C-terminal tail regulates PTEN activity and intracellular trafficking. The dynamical nature of such regulatory events represents a crucial dimension for timing cellular decisions. Here we show that NMR spectroscopy allows reporting on the order and kinetics of clustered multisite phosphorylation events. We first unambiguously identify in vitro seven bona fide sites modified by CK2 and GSK3ß kinases and two new sites on the PTEN C-terminal tail. Then, monitoring the formation of transient intermediate phosphorylated states, we determine the sequence of these reactions and calculate their apparent rate constants. Finally, we assess the dynamic formation of these phosphorylation events induced by endogenous kinases directly in extracts of human neuroblastoma cells. Taken together, our data indicate that two cascades of events controlled by CK2 and GSK3ß occur independently on two clusters of sites (S380-S385 and S361-S370) and that in each cluster the reactions follow an ordered model with a distributive kinetic mechanism. Besides emphasizing the ability of NMR to quantitatively and dynamically follow post-translational modifications, these results bring a temporal dimension on the establishment of PTEN phosphorylation cascades.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosfohidrolasa PTEN/química , FosforilaciónRESUMEN
PTEN (phosphatase and tensin homolog deleted on chromosome 10) and MAST2 (microtubule-associated serine and threonine kinase 2) interact with each other through the PDZ domain of MAST2 (MAST2-PDZ) and the carboxyl-terminal (C-terminal) PDZ domain-binding site (PDZ-BS) of PTEN. These two proteins function as negative regulators of cell survival pathways, and silencing of either one promotes neuronal survival. In human neuroblastoma cells infected with rabies virus (RABV), the C-terminal PDZ domain of the viral glycoprotein (G protein) can target MAST2-PDZ, and RABV infection triggers neuronal survival in a PDZ-BS-dependent fashion. These findings suggest that the PTEN-MAST2 complex inhibits neuronal survival and that viral G protein disrupts this complex through competition with PTEN for binding to MAST2-PDZ. We showed that the C-terminal sequences of PTEN and the viral G protein bound to MAST2-PDZ with similar affinities. Nuclear magnetic resonance structures of these complexes exhibited similar large interaction surfaces, providing a structural basis for their binding specificities. Additionally, the viral G protein promoted the nuclear exclusion of PTEN in infected neuroblastoma cells in a PDZ-BS-dependent manner without altering total PTEN abundance. These findings suggest that formation of the PTEN-MAST2 complex is specifically affected by the viral G protein and emphasize how disruption of a critical protein-protein interaction regulates intracellular PTEN trafficking. In turn, the data show how the viral protein might be used to decipher the underlying molecular mechanisms and to clarify how the subcellular localization of PTEN regulates neuronal survival.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Neuronas/fisiología , Fosfohidrolasa PTEN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Virus de la Rabia/metabolismo , Proteínas Virales/metabolismo , Unión Competitiva , Western Blotting , Calorimetría , Línea Celular Tumoral , Supervivencia Celular/fisiología , Glicoproteínas/química , Humanos , Inmunohistoquímica , Marcaje Isotópico , Proteínas Asociadas a Microtúbulos/química , Neuronas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Dominios PDZ/fisiología , Fosfohidrolasa PTEN/química , Proteínas Serina-Treonina Quinasas/química , Espectrometría de Fluorescencia , Proteínas Virales/químicaRESUMEN
PTPN4, a human tyrosine phosphatase, protects cells against apoptosis. This protection could be abrogated by targeting the PDZ domain of this phosphatase with a peptide mimicking the C-terminal sequence of the G protein of an attenuated rabies virus strain. Here, we demonstrate that glioblastoma death is triggered upon intracellular delivery of peptides, either from viral origin or from known endogenous ligands of PTPN4-PDZ, such as the C terminus sequence of the glutamate receptor subunit GluN2A. The killing efficiency of peptides closely reflects their affinities for the PTPN4-PDZ. The crystal structures of two PTPN4-PDZ/peptide complexes allow us to pinpoint the main structural determinants of binding and to synthesize a peptide of high affinity for PTPN4-PDZ enhancing markedly its cell death capacity. These results allow us to propose a potential mechanism for the efficiency of peptides and provide a target and a robust framework for the design of new pro-death compounds.
Asunto(s)
Muerte Celular , Glioblastoma/patología , Dominios PDZ , Péptidos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 4/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Citometría de Flujo , Glioblastoma/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Virus de la Rabia/química , Receptores de N-Metil-D-Aspartato/metabolismo , Alineación de Secuencia , Proteínas Virales/síntesis química , Proteínas Virales/farmacologíaRESUMEN
Rabies virus (RABV) is a neurotropic virus transmitted by the bite of an infected animal that triggers a fatal encephalomyelitis. During its migration in the nervous system (NS), RABV triggers an innate immune response, including a type I IFN response well known to limit viral infections. We showed that although the neuroinvasive RABV strain CVS-NIV dampens type I IFN signaling by inhibiting IRF3 phosphorylation and STAT2 translocation, an early and transient type I IFN response is still triggered in the infected neuronal cells and NS. This urged us to investigate the role of type I IFN on RABV infection. We showed that primary mouse neurons (DRGs) of type I IFN(α/ß) receptor deficient mice (IFNAR(-/-) mice) were more susceptible to RABV than DRGs of WT mice. In addition, exogenous type I IFN is partially efficient in preventing and slowing down infection in human neuroblastoma cells. Intra-muscular inoculation of type I IFNAR deficient mice [IFNAR(-/-) mice and NesCre ((+/-)) IFNAR ((flox/flox)) mice lacking IFNAR in neural cells of neuroectodermal origin only] with RABV reveals that the type I IFN response limits RABV dissemination in the inoculated muscle, slows down invasion of the spinal cord, and delays mortality. Thus, the type I IFN which is still produced in the NS during RABV infection is efficient enough to reduce neuroinvasiveness and pathogenicity and partially protect the host from fatal infection.