RESUMEN
At present, the mechanism of siSCN9A in Vincristine (VCR)-induced neuropathic pain (NP) is still unclear. This study aimed to explore the analgesic mechanism of lentivirus-siSCN9A (LV-siSCN9A) infected neurons against NP. 40 male Sprague-Dawley (SD) rats were divided into a control group (injected with normal saline), a model group (VCR-induced NP model), a LV-SC group (NP model mice were injected with LV-SC-infected dorsal root ganglia (DRG) neuron cells under the microscope), and a LV-siSCN9A group (NP model mice were injected with LV-siSCN9A-infected DRG neuron cells under the microscope, with 10 rats in each group. The changes of mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats in different groups were detected by behavior testing, the Nav1.7 changes in each group were detected by immunofluorescence double standard and Western-blot method. It was found that compared with the control group, the MWT and TWL of the rats in model group were significantly decreased (P < 0.05), and the expression levels of Nav1.7 messenger ribonucleic acid (mRNA) and proteins were significantly increased (P < 0.05). Compared with LV-SC group, the MWT and TWL of rats in LV-siSCN9A group were significantly increased (P < 0.05), the expression levels of Nav1.7 mRNA and proteins were significantly decreased (P < 0.05), and the CGRP expression of spinal dorsal horn was significantly decreased. It was concluded that the LV-siSCN9A infected neurons could play an analgesic role by down-regulating Nav1.7 expression induced by VCR in NP model.
Asunto(s)
Infecciones por Lentivirus/virología , Lentivirus/patogenicidad , Neuralgia/inducido químicamente , Neuralgia/virología , Neuronas/efectos de los fármacos , Neuronas/virología , Vincristina/farmacología , Analgesia/métodos , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/virología , Infecciones por Lentivirus/genética , Masculino , Ratones , Canal de Sodio Activado por Voltaje NAV1.7/genética , Neuralgia/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity. METHODS: Here, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3+ human T cells. RESULTS: This redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor. CONCLUSIONS: These findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy.
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Anticuerpos Biespecíficos/metabolismo , Lentivirus/patogenicidad , Receptores Quiméricos de Antígenos/metabolismo , Ingeniería de Tejidos/métodos , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Optical spectroscopic techniques have been commonly used to detect the presence of biofilm-forming pathogens (bacteria and fungi) in the agro-food industry. Recently, near-infrared (NIR) spectroscopy revealed that it is also possible to detect the presence of viruses in animal and vegetal tissues. Here we report a platform based on visible and NIR (VNIR) hyperspectral imaging for non-contact, reagent free detection and quantification of laboratory-engineered viral particles in fluid samples (liquid droplets and dry residue) using both partial least square-discriminant analysis and artificial feed-forward neural networks. The detection was successfully achieved in preparations of phosphate buffered solution and artificial saliva, with an equivalent pixel volume of 4 nL and lowest concentration of 800 TU·[Formula: see text]L-1. This method constitutes an innovative approach that could be potentially used at point of care for rapid mass screening of viral infectious diseases and monitoring of the SARS-CoV-2 pandemic.
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Procesamiento de Imagen Asistido por Computador/métodos , Infecciones por Lentivirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Espectroscopía Infrarroja Corta/métodos , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador/normas , Lentivirus/aislamiento & purificación , Lentivirus/patogenicidad , Infecciones por Lentivirus/virología , Técnicas de Diagnóstico Molecular/normas , Sistemas de Atención de Punto , Saliva/virología , Sensibilidad y Especificidad , Espectroscopía Infrarroja Corta/normasRESUMEN
Systemic oxygen restriction (SOR) is prevalent in numerous clinical conditions, including chronic obstructive pulmonary disease (COPD), and is associated with increased susceptibility to viral infections. However, the influence of SOR on T cell immunity remains uncharacterized. Here we show the detrimental effect of hypoxia on mitochondrial-biogenesis in activated mouse CD8+ T cells. We find that low oxygen level diminishes CD8+ T cell anti-viral response in vivo. We reveal that respiratory restriction inhibits ATP-dependent matrix processes that are critical for mitochondrial-biogenesis. This respiratory restriction-mediated effect could be rescued by TCA cycle re-stimulation, which yielded increased mitochondrial matrix-localized ATP via substrate-level phosphorylation. Finally, we demonstrate that the hypoxia-arrested CD8+ T cell anti-viral response could be rescued in vivo through brief exposure to atmospheric oxygen pressure. Overall, these findings elucidate the detrimental effect of hypoxia on mitochondrial-biogenesis in activated CD8+ T cells, and suggest a new approach for reducing viral infections in COPD.
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Adenosina Trifosfato/metabolismo , Linfocitos T CD8-positivos/metabolismo , Hipoxia/metabolismo , Activación de Linfocitos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Biogénesis de Organelos , Fosforilación Oxidativa , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Hipoxia/inmunología , Lentivirus/patogenicidad , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/inmunología , Transducción de SeñalRESUMEN
SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.
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VIH/genética , Lentivirus/genética , Transcripción Reversa/genética , Proteína 1 que Contiene Dominios SAM y HD/genética , Animales , Gatos , Bovinos , Células HEK293 , VIH/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Lentivirus/patogenicidad , Células Mieloides/virología , Dominios Proteicos/genética , Proteína 1 que Contiene Dominios SAM y HD/química , Linfocitos T/virología , Replicación Viral/genéticaRESUMEN
The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson's disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.
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Epigenoma/genética , Epigenómica/métodos , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Lentivirus/patogenicidad , Diferenciación Celular , HumanosRESUMEN
Small ruminant lentivirus (SRLV) infection manifests itself mainly with chronic progressive arthritis affecting mainly carpal joints. The data from serological and questionnaire surveys were retrospectively analyzed to determine how the dissemination of SRLV infection in the herd influenced farmer's subjective opinion on the occurrence of swelling of carpal joints (considered as a proxy of arthritis). Between 1996 and 2017 153 different Polish dairy goat herds counting at least 20 adult goats were serologically screened for CAE and their owners were asked about their opinion on the occurrence of arthritis (never, rarely, often). Of them 73 SRLV-seropositive herds, in which true seroprevalence had been estimated, were included in the analysis. The ordinal logistic regression model was developed to determine the relationship between the true within-herd seroprevalence and the probability that the farmer would observe arthritis in the herd never, rarely or often. True within-herd seroprevalence ranged from 0.2% to 100% with the median of 34.6%. Farmers declared not to have observed arthritis in 40 (54.8%) herds, to have seen it rarely in 9 (12.3%) of herds, and to have observed it often in 24 (32.9%) of herds. The model proved that the probability of observing goats with carpal arthritis in the herd was significantly linked to the true within-herd seroprevalence (OR = 1.058, CI 95% from 1.037 to 1.078; p<0.001), but this relationship was not linear and SRLV infection proved to remain unapparent to farmers even when a considerable part of the herd had already become infected. Concluding, the study shows that when the farmer realizes that goats in the herd suffer from arthritis, SRLV infection is almost certainly already widespread in the herd.
Asunto(s)
Anticuerpos Antivirales/sangre , Artritis/veterinaria , Articulaciones del Carpo/virología , Agricultores/psicología , Enfermedades de las Cabras/epidemiología , Cabras/virología , Infecciones por Lentivirus/veterinaria , Crianza de Animales Domésticos , Animales , Artritis/epidemiología , Artritis/patología , Artritis/virología , Articulaciones del Carpo/inmunología , Articulaciones del Carpo/patología , Femenino , Enfermedades de las Cabras/inmunología , Enfermedades de las Cabras/patología , Enfermedades de las Cabras/virología , Lentivirus/patogenicidad , Lentivirus/fisiología , Infecciones por Lentivirus/epidemiología , Infecciones por Lentivirus/patología , Infecciones por Lentivirus/virología , Modelos Logísticos , Masculino , Polonia/epidemiología , Estudios Seroepidemiológicos , Encuestas y CuestionariosRESUMEN
Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis. Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status.
Asunto(s)
Enfermedades de las Cabras/patología , Enfermedades de las Cabras/virología , Infecciones por Lentivirus/veterinaria , Animales , Genotipo , Cabras , Lentivirus/aislamiento & purificación , Lentivirus/patogenicidad , Infecciones por Lentivirus/patología , Infecciones por Lentivirus/virología , Rumiantes , Ovinos , Enfermedades de las Ovejas , Distribución Tisular , Carga Viral/veterinariaRESUMEN
BACKGROUND: shRNA lentiviral vectors are extensively used for gene knockdowns in mammalian cells, and non-target shRNAs typically are considered the proper experimental control for general changes caused by RNAi. However, the effects of non-target lentivirus controls on the modulation of cell signaling pathways remain largely unknown. In this study, we evaluated the effect of control lentiviral transduction on oxytocin receptor (OXTR) expression through the ERK/MAPK pathway in mouse and human skeletal muscle cells, on myogenic activity, and in vivo on mouse muscle regeneration. Furthermore, we mined published data for the influence of viral infections on OXTR levels in human populations and found that unrelated viral pathologies have a common consequence: diminished levels of OXTR. METHODS: We examined the change in OXTR mRNA expression upon transduction with control and Smad3-targeting viral vectors through real time RT-PCR and Western blotting, and confirmed with immunofluorescence. Changes in Smad3 and OXTR expression were examined both in vitro with mouse and human myoblasts and in vivo in mouse satellite cells. The general effects of viral infections on OXTR downregulation in humans were also examined by analyzing published Gene Expression Omnibus (GEO) datasets. The change in myoblast myogenic activity caused by the viral transduction (the percent of Pax7 + Ki67+ cells) was examined by immunofluorescence. RESULTS: Results shown in this work establish that lentiviral control vectors significantly downregulate OXTR expression at mRNA and protein levels and diminish key downstream effectors of OXTR, ERK signaling, reducing the myogenic proliferation of infected cells. This effect is evolutionarily conserved between mouse and human myogenic cells, and it manifests in satellite cells after control lentiviral transduction of mice in vivo. Furthermore, an examination of published datasets uncovered similar OXTR downregulation in humans that are afflicted with different viral infections. Additionally, cells transduced with Smad3-targeting shRNA downregulate OXTR even more than cells transduced with control viruses. CONCLUSIONS: Our work suggests that experimental cohorts transduced with control viruses may not behave the same as un-transduced cells and animals, specifically that control viral vectors significantly change the intensity of key cell-signaling pathways, such as OXTR/ERK. Our results further demonstrate that lentiviral transduction significantly decreases myogenic proliferation and suggest that viral infections in general may play a role in decreasing muscle health and regeneration, a decline in metabolic health, and a lower sense of well-being, as these rely on effective OXTR signaling. Additionally, our data suggest pathway crosstalk between TGF-ß/pSmad3 and OXTR, implying that sustained attenuation of the TGF-ß/pSmad3 pathway will reduce pro-regenerative OXTR/pERK signaling.
Asunto(s)
Receptores de Oxitocina/genética , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Virosis/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Evolución Molecular , Humanos , Lentivirus/patogenicidad , Ratones , Ratones Endogámicos C57BL , Mioblastos/metabolismo , Mioblastos/virología , Receptores de Oxitocina/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/virología , Virosis/genéticaRESUMEN
In nature, primate lentiviruses infect humans and several Old World monkeys and apes. However, to date, lentiviruses infecting New World monkeys have not been described. We studied the susceptibility of common marmoset cells to HIV-1 infection and observed the presence of post-entry blocks to the early phase of HIV-1 infection in peripheral blood lymphocytes (PBLs) and a B lymphocytic cell line (B-LCL). The blocks present in these cells are dominant and phenotypically different from each other. In PBLs, the block occurs at the level of reverse transcription, reducing the accumulation of early and late transcripts, similar to the block imposed by TRIM5α. However, we have found that marmoset TRIM5α does not block HIV-1. In contrast, the restriction factor present in B-LCLs blocks HIV-1 replication at a later step, after nuclear entry, and inhibits integration. Additionally, we have identified an HIV-1 capsid mutant, N74D, that is able to escape the restriction in the marmoset B-LCLs. Our results suggest that the factors responsible for the blocks present in marmoset PBLs and B-LCLs are different. We propose the existence of at least two new restriction factors able to block HIV-1 infection in marmoset lymphocytes.
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Proteínas de la Cápside/genética , Infecciones por VIH/virología , VIH-1/genética , Lentivirus/genética , Animales , Factores de Restricción Antivirales , Linfocitos B/inmunología , Linfocitos B/virología , Callithrix/inmunología , Callithrix/virología , Proteínas de la Cápside/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Lentivirus/inmunología , Lentivirus/patogenicidad , Linfocitos/inmunología , Linfocitos/virología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Internalización del Virus , Replicación Viral/genéticaRESUMEN
UNLABELLED: Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE: Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.
Asunto(s)
Infecciones por Arterivirus/epidemiología , Evolución Biológica , Infecciones por Flaviviridae/epidemiología , Variación Genética , Infecciones por Lentivirus/epidemiología , Carga Viral , África/epidemiología , Animales , Animales Salvajes , Arterivirus/genética , Arterivirus/patogenicidad , Infecciones por Arterivirus/genética , Infecciones por Arterivirus/virología , Flaviviridae/genética , Flaviviridae/patogenicidad , Infecciones por Flaviviridae/genética , Infecciones por Flaviviridae/virología , Genoma Viral , Haplorrinos , Humanos , Lentivirus/genética , Lentivirus/patogenicidad , Infecciones por Lentivirus/genética , Infecciones por Lentivirus/virología , Filogenia , PrevalenciaRESUMEN
Pathogens such as HIV-1, with their minimalist genomes, must navigate cellular networks and rely on hijacking and manipulating the host machinery for successful replication. Limited overlap of host factors identified as vital for pathogen replication may be explained by considering that pathogens target, rather than specific cellular factors, crucial cellular pathways by targeting different, functionally equivalent, protein-protein interactions within that pathway. The ability to utilize alternative routes through cellular pathways may be essential for pathogen survival when restricted and provide flexibility depending on the viral replication stage and the environment in the infected host. In this minireview, we evaluate evidence supporting this notion, discuss specific HIV-1 examples, and consider the molecular mechanisms which allow pathogens to flexibly exploit different routes.
Asunto(s)
Infecciones por VIH/virología , VIH-1/patogenicidad , Transporte Activo de Núcleo Celular , Animales , Citidina Desaminasa/fisiología , VIH-1/genética , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Lentivirus/patogenicidad , Lentivirus/fisiología , Infecciones por Lentivirus/virología , Modelos Biológicos , Integración Viral , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
To achieve productive infection, retroviruses such as HIV stably integrate their reverse transcribed RNA genome into a host chromosome. Each retroviral family preferentially integrates near a unique subset of genomic features. HIV integrase (IN) is targeted to the body of active transcription units through interaction with lens epithelium-derived growth factor (LEDGF/p75). We describe the successful effort to develop inhibitors of the interaction between IN and LEDGF/p75, referred to as LEDGINs. Gammaretroviruses display a distinct integration pattern. Recently, BET (bromo- and extraterminal domain) proteins were identified as the LEDGF/p75 counterparts that target the integration of gammaretroviruses. The identification of the chromatin-readers LEDGF/p75 and BET as cellular cofactors that orchestrate lentiviral or gammaretroviral integration opens new avenues to developing safer viral vectors for gene therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/administración & dosificación , Factores de Transcripción/metabolismo , Integración Viral/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Cromatina/efectos de los fármacos , Gammaretrovirus/efectos de los fármacos , Gammaretrovirus/genética , Gammaretrovirus/patogenicidad , Infecciones por VIH/virología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Lentivirus/efectos de los fármacos , Lentivirus/genética , Lentivirus/patogenicidad , Factores de Transcripción/genética , Integración Viral/efectos de los fármacosRESUMEN
Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins; however, its physiological roles remain unknown. Here we identify MARCH8 as a novel antiviral factor. The overexpression of MARCH8 in virus producing cells did not affect levels of lentivirus production, but markedly reduced viral infectivity. MARCH8 blocked the incorporation of HIV-1 envelope glycoprotein into virions by downregulating it from the cell surface, probably through their interaction, resulting in reduced viral entry efficiency. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein was even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Importantly, the endogenous expression of MARCH8 was high in monocyte-derived macrophages and dendritic cells, and MARCH8 depletion in macrophages significantly increased the infectivity of virions produced from these cells. Our findings thus indicate that MARCH8, which is highly expressed in terminally differentiated myeloid cells, is a potent antiviral host transmembrane protein that reduces virion incorporation of viral envelope glycoproteins.
Asunto(s)
Antivirales , VIH-1/patogenicidad , Lentivirus/patogenicidad , Ubiquitina-Proteína Ligasas/fisiología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Glicoproteínas/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Glicoproteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Células Mieloides/virología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismoRESUMEN
Infection is best described as a stochastic process. Whether a host becomes infected upon exposure has a strong random element. The same applies to cells exposed to virions. In this review, we show how the mathematical formalism for stochastic processes has been used to describe and understand the infection by the Human and Simian Immunodeficiency Virus on different levels. We survey quantitative studies on the establishment of infection in the host (the organismal level) and on the infection of target cells (the cellular and molecular level). We then discuss how a synthesis of the approaches across these levels could give rise to a predictive framework for assessing the efficacy of microbicides and vaccines.
Asunto(s)
Interacciones Huésped-Patógeno , Infecciones por Lentivirus/virología , Lentivirus/química , Lentivirus/patogenicidad , Primates/virología , Subunidades de Proteína/metabolismo , Animales , Humanos , Internalización del VirusRESUMEN
Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.
Asunto(s)
Carcinogénesis/genética , Terapia Genética , Vectores Genéticos/efectos adversos , Lentivirus/genética , Vectores Genéticos/uso terapéutico , Humanos , Lentivirus/patogenicidad , Mutagénesis Insercional/genética , Regiones Promotoras GenéticasRESUMEN
Small ruminant lentiviruses (SRLV) are members of the Retrovirus family comprising the closely related Visna/Maedi Virus (VMV) and the Caprine Arthritis-Encephalitis Virus (CAEV), which infect sheep and goats. Both infect cells of the monocyte/macrophage lineage and cause lifelong infections. Infection by VMV and CAEV can lead to Visna/Maedi (VM) and Caprine Arthritis-Encephalitis (CAE) respectively, slow progressive inflammatory diseases primarily affecting the lungs, nervous system, joints and mammary glands. VM and CAE are distributed worldwide and develop over a period of months or years, always leading to the death of the host, with the consequent economic and welfare implications. Currently, the control of VM and CAE relies on the control of transmission and culling of infected animals. However, there is evidence that host genetics play an important role in determining Susceptibility/Resistance to SRLV infection and disease progression, but little work has been performed in small ruminants. More research is necessary to understand the host-SRLV interaction.
Asunto(s)
Enfermedades de las Cabras/virología , Interacciones Huésped-Patógeno , Infecciones por Lentivirus/veterinaria , Lentivirus/patogenicidad , Enfermedades de las Ovejas/virología , Animales , Enfermedades de las Cabras/patología , Enfermedades de las Cabras/prevención & control , Enfermedades de las Cabras/transmisión , Cabras , Infecciones por Lentivirus/patología , Infecciones por Lentivirus/transmisión , Infecciones por Lentivirus/virología , Ovinos , Enfermedades de las Ovejas/patología , Enfermedades de las Ovejas/prevención & control , Enfermedades de las Ovejas/transmisiónRESUMEN
Objectives KLF8 is a member of KLF transcription factors which play an important tolr in oncogenesis. It is barely expressed in normal human epithelial cells but highly overexpressed in several types of human cancer cell lines. In the present study, we investigate the role of KLF8 in oral cancer and the effects of KLF8 knockdown via lentivirus mediated siRNA infection in human adenosquamos carcinoma CAL 27 cells. Study Design We developed a vector-based siRNA expression system that can induce RNAi in CAL 27 oral cancer cells. Downregulation of KLF8 was confirmed by evaluating GFP expressions, RT-PCR and western blot analysis. Finally, the effects of KLF8 downregulation were analyzed by MTT assay and colony formation assays. Results The expression levels of KLF8 mRNA and proteins are reduced in CAL 27 cells that transfected with 21-nt siRNA against KLF8. Lentivirus-mediated silencing of KLF8 reduces cell proliferation and colonies number, thereby indicating the role of KLF8 in cell proliferation and tumorigenesis. Conclusions These results strongly suggest that KLF8 is essential for growth of CAL 27 cancer cells. A better understanding of KLF8 function and processing may provide novel insights into the clinical therapy of oral cancer (AU)
Asunto(s)
Humanos , Factores de Transcripción de Tipo Kruppel/análisis , Neoplasias de la Boca/patología , Lentivirus/patogenicidad , Proliferación Celular , Receptor Xedar/análisisRESUMEN
Rhesus macaque APOBEC3A (rhA3A) is capable of restricting both simian-human immunodeficiency virus (SHIVΔvif) and human immunodeficiency virus (HIV-1Δvif) to a greater extent than hA3A. We constructed chimeric A3A proteins to define the domains required for differential lentivirus restriction. Substitution of amino acids 25-33 from rhA3A into hA3A was sufficient to restrict HIVΔvif to levels similar to rhA3A restriction of SHIVΔvif. We tested if differential lentivirus restriction is conserved between A3A from Old World monkey and hominid lineages. A3A from African green monkey restricted SHIVΔvif but not HIV-1Δvif and colobus monkey A3A restricted both wild type and SHIVΔvif and HIV-1Δvif. In contrast, the gibbon ape A3A restricted neither SHIVΔvif nor HIV-1Δvif. Restriction of SHIVΔvif and HIV-1Δvif by New World monkey A3A proteins was not conserved as the A3A from the squirrel monkey but not the northern owl monkey restricted SHIVΔvif. Finally, the colobus A3A protein appears to restrict by a novel post-entry mechanism.
Asunto(s)
Citidina Desaminasa/metabolismo , VIH-1/patogenicidad , Primates/virología , Proteínas/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Replicación Viral , Animales , Línea Celular , Chlorocebus aethiops , Citidina Desaminasa/genética , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Lentivirus/patogenicidad , Lentivirus/fisiología , Macaca mulatta/virología , Proteínas/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/metabolismo , Virus de la Inmunodeficiencia de los Simios/fisiologíaRESUMEN
α-Melanocyte-stimulating hormone (α-MSH) functions as a mediator of inflammation and immunity; however, the short half-life and high dose needed limit the comprehensive clinical application of α-MSH. The aim of this study was to generate human bone marrow-derived mesenchymal stem cells (MSCs) that express and secrete high levels of bioactive α-MSH. MSCs were obtained from a normal donor and assessed for proliferation, surface markers, and adipogenic and osteogenic differentiation. A lentivirus-encoding α-MSH was constructed. MSCs were infected with this lentivirus-encoding α-MSH and assessed for stability and the expression and secretion of bioactive α-MSH. The cumulative MSC expansion rates pre- and post-lentivirus infection were not significantly different (P > 0.05). The MSCs remained stable after infection with the lentivirus-encoding α-MSH. The concentration of α-MSH in the supernatants of MSCs infected with the lentivirus-encoding α-MSH was 17.55 ng/ml (P < 0.001), and a melanin assay indicated that bioactive α-MSH was secreted from MSCs infected with the lentivirus-encoding α-MSH, with an optical absorbance at OD(405) of 0.886 (P < 0.001). These results suggested that MSCs were promising cell carriers for the expression and secretion of high levels of bioactive α-MSH.