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1.
J Neuroinflammation ; 12: 185, 2015 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-26423139

RESUMEN

BACKGROUND: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO. METHODS: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining. RESULTS: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957. CONCLUSIONS: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.


Asunto(s)
Granulocitos/efectos de los fármacos , Inmunoglobulina G/farmacología , FN-kappa B/metabolismo , Neuroglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Antineoplásicos/farmacología , Bortezomib/farmacología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Neuromielitis Óptica/sangre , Neuromielitis Óptica/inmunología , Infiltración Neutrófila/efectos de los fármacos , Oligopéptidos/farmacología , Inhibidores de Proteasoma/farmacología
2.
Bio Protoc ; 10(14): e3679, 2020 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-33659350

RESUMEN

Data generated by metagenomic and metatranscriptomic experiments is both enormous and inherently noisy. When using taxonomy-dependent alignment-based methods to classify and label reads, the first step consists in performing homology searches against sequence databases. To obtain the most information from the samples, nucleotide sequences are usually compared to various databases (nucleotide and protein) using local sequence aligners such as BLASTN and BLASTX. Nevertheless, the analysis and integration of these results can be problematic because the outputs from these searches usually show inconsistencies, which can be notorious when working with RNA-seq. Moreover, and to the best of our knowledge, existing tools do not criss-cross and integrate information from the different homology searches, but provide the results of each analysis separately. We developed the HoSeIn workflow to intersect the information from these homology searches, and then determine the taxonomic and functional profile of the sample using this integrated information. The workflow is based on the assumption that the sequences that correspond to a certain taxon are composed of: sequences that were assigned to the same taxon by both homology searches; sequences that were assigned to that taxon by one of the homology searches but returned no hits in the other one.

3.
Aging (Albany NY) ; 12(14): 15134-15156, 2020 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-32640422

RESUMEN

Multiple sclerosis (MS) is a central nervous system inflammatory demyelinating disease and the most common cause of non-traumatic disability in young adults. Despite progress in the treatment of the active relapsing disease, therapeutic options targeting irreversible progressive decline remain limited. Studies using skin fibroblasts derived from patients with neurodegenerative disorders demonstrate that cell stress pathways and bioenergetics are altered when compared to healthy individuals. However, findings in MS skin fibroblasts are limited. Here, we collected skin fibroblasts from 24 healthy control individuals, 30 patients with MS, and ten with amyotrophic lateral sclerosis (ALS) to investigate altered cell stress profiles. We observed endoplasmic reticulum swelling in MS skin fibroblasts, and increased gene expression of cell stress markers including BIP, ATF4, CHOP, GRP94, P53, and P21. When challenged against hydrogen peroxide, MS skin fibroblasts had reduced resiliency compared to ALS and controls. Mitochondrial and glycolytic functions were perturbed in MS skin fibroblasts while exhibiting a significant increase in lactate production over ALS and controls. Our results suggest that MS skin fibroblasts have an underlying stress phenotype, which may be disease specific. Interrogating MS skin fibroblasts may provide patient specific molecular insights and aid in prognosis, diagnosis, and therapeutic testing enhancing individualized medicine.


Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Esclerosis Amiotrófica Lateral , Retículo Endoplásmico , Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Esclerosis Múltiple , Factor de Transcripción CHOP/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Variación Biológica Poblacional , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Metabolismo Energético/fisiología , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/metabolismo , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Medicina de Precisión , Piel/patología
4.
Methods Protoc ; 2(2)2019 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-31164615

RESUMEN

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

5.
JCI Insight ; 4(18)2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31534057

RESUMEN

Insulin resistance associates with increased risk for cognitive decline and dementia; however, the underpinning mechanisms for this increased risk remain to be fully defined. As insulin resistance impairs mitochondrial oxidative metabolism and increases ROS in skeletal muscle, we considered whether similar events occur in the brain, which - like muscle - is rich in insulin receptors and mitochondria. We show that high-fat diet-induced (HFD-induced) brain insulin resistance in mice decreased mitochondrial ATP production rate and oxidative enzyme activities in brain regions rich in insulin receptors. HFD increased ROS emission and reduced antioxidant enzyme activities, with the concurrent accumulation of oxidatively damaged mitochondrial proteins and increased mitochondrial fission. Improvement of insulin sensitivity by both aerobic exercise and metformin ameliorated HFD-induced abnormalities. Moreover, insulin-induced enhancement of ATP production in primary cortical neurons and astrocytes was counteracted by the insulin receptor antagonist S961, demonstrating a direct effect of insulin resistance on brain mitochondria. Further, intranasal S961 administration prevented exercise-induced improvements in ATP production and ROS emission during HFD, supporting that exercise enhances brain mitochondrial function by improving insulin action. These results support that insulin sensitizing by exercise and metformin restores brain mitochondrial function in insulin-resistant states.


Asunto(s)
Corteza Cerebral/efectos de los fármacos , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Metformina/administración & dosificación , Mitocondrias/efectos de los fármacos , Condicionamiento Físico Animal/fisiología , Receptor de Insulina/metabolismo , Administración Intranasal , Administración Oral , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/patología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/patología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Glucosa/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Dinámicas Mitocondriales/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Péptidos/administración & dosificación , Cultivo Primario de Células , Receptor de Insulina/antagonistas & inhibidores , Conducta Sedentaria
6.
J Virol Methods ; 148(1-2): 286-90, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18242721

RESUMEN

There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.


Asunto(s)
Baculoviridae/genética , Clonación Molecular/métodos , ADN Viral/genética , ADN Viral/aislamiento & purificación , Animales , Línea Celular , Escherichia coli , Lepidópteros , Recombinación Genética , Reproducibilidad de los Resultados
7.
Sci Rep ; 7(1): 9037, 2017 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-28831096

RESUMEN

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1ß, and IFNγ.


Asunto(s)
Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/patología , Citocinas/metabolismo , Imagen Molecular , Vías Nerviosas , Neuronas/fisiología , Animales , Biomarcadores , Señalización del Calcio , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Mediadores de Inflamación/metabolismo , Ratones , Red Nerviosa , Neuronas/efectos de los fármacos , Receptores de Neurotransmisores/agonistas , Receptores de Neurotransmisores/antagonistas & inhibidores
9.
Genome Announc ; 3(4)2015 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-26272581

RESUMEN

This is the first study to use a high-throughput metagenomic shotgun approach to explore the biosynthetic potential of soil metagenomes from different pristine environments of northwest Argentina. Our data sets characterize these metagenomes and provide information on the possible effect these ecosystems have on their diversity and biosynthetic potential.

10.
Genome Announc ; 3(4)2015 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-26184938

RESUMEN

This is the first study to report a high-throughput approach integrating gene expression data from Spodoptera frugiperda guts and their associated metatranscriptomes. Our datasets provide information on the potential effects of environmental conditions on the expression profile of S. frugiperda larval guts, their associated metatranscriptome, and putative interactions between them.

11.
Viruses ; 7(4): 1599-612, 2015 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-25835531

RESUMEN

Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterial ß-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.


Asunto(s)
Entomología/métodos , Lepidópteros/virología , Biología Molecular/métodos , Nucleopoliedrovirus/crecimiento & desarrollo , Nucleopoliedrovirus/genética , Recombinación Genética , Virología/métodos , Animales , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Larva/virología , América del Sur
12.
J Vector Ecol ; 39(1): 83-93, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24820560

RESUMEN

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host-specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).


Asunto(s)
Insectos Vectores/parasitología , Leishmaniasis Visceral/transmisión , Phlebotomus/parasitología , Reacción en Cadena de la Polimerasa/métodos , Psychodidae/parasitología , Animales , Argentina , Femenino
13.
PLoS One ; 8(12): e84020, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24376783

RESUMEN

Gene expression is subject to stochastic variation which leads to fluctuations in the rate of protein production. Recently, a study in yeast at a genomic scale showed that, in some cases, gene expression variability alters phenotypes while, in other cases, these remain unchanged despite fluctuations in the expression of other genes. These studies suggested that noise in gene expression is a physiologically relevant trait and, to prevent harmful stochastic variation in the expression levels of some genes, it can be subject to minimisation. However, the mechanisms for noise minimisation are still unclear. In the present work, we analysed how noise expression depends on the architecture of the cis-regulatory system, in particular on the number of regulatory binding sites. Using analytical calculations and stochastic simulations, we found that the fluctuation level in noise expression decreased with the number of regulatory sites when regulatory transcription factors interacted with only one other bound transcription factor. In contrast, we observed that there was an optimal number of binding sites when transcription factors interacted with many bound transcription factors. This finding suggested a new mechanism for preventing large fluctuations in the expression of genes which are sensitive to the concentration of regulators.


Asunto(s)
Regulación de la Expresión Génica , Modelos Genéticos , Sitios de Unión , Evolución Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Procesos Estocásticos , Factores de Transcripción/metabolismo
14.
PLoS One ; 8(3): e58645, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23554910

RESUMEN

Leishmaniasis is a vector-borne disease with a complex epidemiology and ecology. Visceral leishmaniasis (VL) is its most severe clinical form as it results in death if not treated. In Latin America VL is caused by the protist parasite Leishmania infantum (syn. chagasi) and transmitted by Lutzomyia longipalpis. This phlebotomine sand fly is only found in the New World, from Mexico to Argentina. However, due to deforestation, migration and urbanisation, among others, VL in Latin America is undergoing an evident geographic expansion as well as dramatic changes in its transmission patterns. In this context, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases. Insect vector transcriptomic analyses enable the identification of molecules involved in the insect's biology and vector-parasite interaction. Previous studies on laboratory reared Lu. longipalpis have provided a descriptive repertoire of gene expression in the whole insect, midgut, salivary gland and male reproductive organs. Nevertheless, the study of wild specimens would contribute a unique insight into the development of novel bioinsecticides. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. In this study, total RNA was extracted from the sand flies, submitted to sequence independent amplification and high-throughput pyrosequencing. This is the first time an unbiased and comprehensive transcriptomic approach has been used to analyse an infectious disease vector in its natural environment. Transcripts identified in the sand flies showed characteristic profiles which correlated with the environment of origin and with taxa previously identified in these same specimens. Among these, various genes represented putative targets for vector control via RNA interference (RNAi).


Asunto(s)
Insectos Vectores/metabolismo , Leishmania infantum , Leishmaniasis Visceral/transmisión , Psychodidae/metabolismo , ARN/biosíntesis , Transcriptoma , Animales , Argentina/epidemiología , Brotes de Enfermedades , Femenino , Humanos , Insectos Vectores/genética , Insectos Vectores/parasitología , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/parasitología , Masculino , Psychodidae/genética , Psychodidae/parasitología , ARN/genética
15.
PLoS Negl Trop Dis ; 5(9): e1304, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21909446

RESUMEN

BACKGROUND: Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases. METHODOLOGY/PRINCIPAL FINDINGS: An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans. CONCLUSIONS/SIGNIFICANCE: This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.


Asunto(s)
Biodiversidad , Vectores de Enfermedades , Metagenoma/genética , Psychodidae/parasitología , Animales , Argentina , Brasil , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , América Latina , Masculino , Plantas/genética , ARN/genética , ARN/aislamiento & purificación
16.
Virology ; 375(1): 277-91, 2008 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-18328526

RESUMEN

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC(ac142)(REP-ac143KO)). Fluorescence and light microscopy showed that infection by AcBAC(ac142)(REP-ac143KO) is limited to a single cell and titration assays confirmed that AcBAC(ac142)(REP-ac143KO) was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC(ac142)(REP-ac143KO) transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.


Asunto(s)
Nucleopoliedrovirus/fisiología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus/genética , Replicación Viral/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/virología , Eliminación de Gen , Orden Génico , Genes Esenciales , Cuerpos de Inclusión Intranucleares/virología , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Regiones Promotoras Genéticas , Alineación de Secuencia , Eliminación de Secuencia , Spodoptera
17.
Virology ; 372(2): 325-39, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18045640

RESUMEN

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC(ac142KO-PH-GFP)). Fluorescence and light microscopy revealed that AcBAC(ac142KO-PH-GFP) exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC(ac142KO-PH-GFP) is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC(ac142KO-PH-GFP)-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.


Asunto(s)
Genes Esenciales/genética , Genes Virales/genética , Nucleopoliedrovirus/crecimiento & desarrollo , Nucleopoliedrovirus/genética , Proteínas Virales/metabolismo , Replicación Viral/genética , Animales , Línea Celular , Eliminación de Gen , Regulación Viral de la Expresión Génica , Nucleocápside/ultraestructura , Nucleopoliedrovirus/ultraestructura , Spodoptera/citología , Transcripción Genética , Proteínas Virales/genética , Virosis
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