RESUMEN
Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1ß. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Pez Cebra , Animales , Pez Cebra/virología , Rhabdoviridae/fisiología , Enfermedades de los Peces/virología , Infecciones por Rhabdoviridae/virología , Infecciones por Rhabdoviridae/inmunología , Carpas/virología , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Inmunidad Innata , ViremiaRESUMEN
The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.
Asunto(s)
Septicemia Hemorrágica Viral , Nodaviridae , Novirhabdovirus , Vacunas , Animales , Nodaviridae/genética , Glicoproteínas , AntígenosRESUMEN
Salmonid alphavirus (SAV) is an atypical alphavirus that has a considerable impact on salmon and trout farms. Unlike other alphaviruses, such as the chikungunya virus, SAV is transmitted without an arthropod vector, and it does not cause cell shutoff during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that nonstructural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3, which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus, which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell's innate immune response. IMPORTANCE The global consumption of fish continues to rise, and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world's fastest-growing food production sector, with an annual growth rate of 6 to 8%. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences for wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the nonstructural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.
Asunto(s)
Alphavirus/metabolismo , Antivirales/farmacología , Proteína 58 DEAD Box/metabolismo , Interferones/metabolismo , Salmonidae/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Línea Celular , Virus Chikungunya , Enfermedades de los Peces/virología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Inmunidad Innata , Interferón Tipo I/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Interferons are the first lines of defense against viral pathogen invasion during the early stages of infection. Their synthesis is tightly regulated to prevent excessive immune responses and possible deleterious effects on the host organism itself. The RIG-I-like receptor signaling cascade is one of the major pathways leading to the production of interferons. This pathway amplifies danger signals and mounts an appropriate innate response but also needs to be finely regulated to allow a rapid return to immune homeostasis. Recent advances have characterized different cellular factors involved in the control of the RIG-I pathway. This has been most extensively studied in mammalian species; however, some inconsistencies remain to be resolved. The IFN system is remarkably well conserved in vertebrates and teleost fish possess all functional orthologs of mammalian RIG-I-like receptors as well as most downstream signaling molecules. Orthologs of almost all mammalian regulatory components described to date exist in teleost fish, such as the widely used zebrafish, making fish attractive and powerful models to study in detail the regulation and evolution of the RIG-I pathway.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Peces/genética , Peces/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras , Proteína 58 DEAD Box/genética , Peces/inmunología , Regulación de la Expresión Génica , Homeostasis , Inmunidad Innata , Interferones/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Unión Proteica , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state.
Asunto(s)
Cyprinidae/genética , Cyprinidae/inmunología , Proteína 58 DEAD Box/genética , Enfermedades de los Peces/inmunología , Inmunidad Innata/genética , Interferones/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Animales , Línea Celular , Proteína 58 DEAD Box/metabolismo , Retroalimentación Fisiológica , Proteínas de Peces/genética , Proteínas de Peces/fisiología , Interferones/metabolismo , Novirhabdovirus/fisiología , Filogenia , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
Asunto(s)
Neoplasias/genética , Neoplasias/patología , Interferencia de ARN , Línea Celular Tumoral , Biblioteca de Genes , Redes Reguladoras de Genes , Humanos , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.
Asunto(s)
Enfermedades de los Peces/metabolismo , Proteínas de Peces/metabolismo , Interferones/metabolismo , Novirhabdovirus/metabolismo , Oncorhynchus mykiss/metabolismo , Proteína Fosfatasa 2C/metabolismo , Infecciones por Rhabdoviridae/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Novirhabdovirus/genética , Oncorhynchus mykiss/virología , Infecciones por Rhabdoviridae/genética , Proteínas Virales/genéticaRESUMEN
The synthesis of o-boronato- and o-trifluoroborato-phosphonium salts supported by the L-amino acid side chain is described. The synthesis of these new class of amino acid derivatives was achieved by stereoselective quaternization of o-(pinacolato)boronatophenylphosphine with ß- or γ-iodo amino acid derivatives which are prepared from L-serine or L-aspartic acid, respectively. The quaternization of the phosphine was performed using either iodo amino ester or carboxylic acid derivatives. In addition, free carboxylic acid and amine derivatives were obtained by saponification or HCl acidolysis of o-boronato-phosphonium amino esters, respectively. The usefulness of these compounds in peptide coupling was demonstrated by coupling an o-boronato-phosphonium amino ester with an aspartic acid moiety. When the o-boronato-phosphonium amino acid or dipeptide derivatives were mixed with fluoride, the corresponding o-trifluoroborated products were cleanly and rapidly obtained in high isolated yields. The hydrolysis of these compounds at room temperature using a phosphate buffer pH 7/CD3CN mixture has shown only traces of free fluoride F(-) after several days. Finally, a preliminary radiolabeling essay has proven the facile [(18)F]-fluoride incorporation and high stability of the radiolabeled product in aqueous conditions. Indeed, this new class of boron-phosphonium amino acid derivatives shows promising properties for their applications in synthesis and labeling of peptides.
Asunto(s)
Aminoácidos/química , Boratos/síntesis química , Compuestos Organofosforados/síntesis química , Boratos/química , Estructura Molecular , Compuestos Organofosforados/química , Sales (Química)/síntesis química , Sales (Química)/químicaRESUMEN
An efficient synthesis of boronated phosphines with an o-phenylene-bridge prepared from sec-phosphine boranes and using benzyne chemistry is reported. Successive reactions of sec-phosphine boranes with n-BuLi and 1,2-dibromobenzene, and then with boron reagents, afford the o-boronatophenylphosphine derivatives in 71% yields. The use of P-chirogenic sec-phosphine boranes leads to the free boronated phosphines with retention of configuration at the P-center after decomplexation. The reaction of P-chirogenic o-boronatophenylphosphine with KHF2 affords the corresponding trifluoroborated phosphine with ee >98%.
RESUMEN
A recombinant sleeping disease virus (rSDV) was previously shown to be totally attenuated and provide long-term protection in trout (C. Moriette, M. Leberre, A. Lamoureux, T. L. Lai, M. Brémont, J. Virol. 80:4088-4098, 2006). Sequence comparison of the rSDV to wild-type genomes exhibited a number of nucleotide changes. In the current study, we demonstrate that the virulent phenotype of SDV was essentially associated with two amino acid changes, V8A and M136T, in the E2 glycoprotein, with the V8A change mostly being involved in the acquisition of the virulent phenotype.
Asunto(s)
Alphavirus/genética , Alphavirus/patogenicidad , Sustitución de Aminoácidos , Glicoproteínas/genética , Mutación Missense , Proteínas Virales/genética , Factores de Virulencia/genética , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Enfermedades de los Peces/virología , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Análisis de Supervivencia , Trucha , Proteínas Virales/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Viral infections are detected in most cases by the host innate immune system through pattern-recognition receptors (PRR), the sensors for pathogen-associated molecular patterns (PAMPs), which induce the production of cytokines, such as type I interferons (IFN). Recent identification in mammalian and teleost fish of cytoplasmic viral RNA sensors, RIG-I-like receptors (RLRs), and their mitochondrial adaptor: the mitochondrial antiviral signaling (MAVS) protein, also called IPS-1, highlight their important role in the induction of IFN at the early stage of a virus infection. More recently, an endoplasmic reticulum (ER) adaptor: the stimulator of interferon genes (STING) protein, also called MITA, ERIS and MPYS, has been shown to play a pivotal role in response to both non-self-cytosolic RNA and dsDNA. In this study, we cloned STING cDNAs from zebrafish and showed that it was an ortholog to mammalian STING. We demonstrated that overexpression of this ER protein in fish cells led to a constitutive induction of IFN and interferon-stimulated genes (ISGs). STING-overexpressing cells were almost fully protected against RNA virus infection with a strong inhibition of both DNA and RNA virus replication. In addition, we found that together with MAVS, STING was an important player in the RIG-I IFN-inducing pathway. This report provides the demonstration that teleost fish possess a functional RLR pathway in which MAVS and STING are downstream signaling molecules of RIG-I. The Sequences presented in this article have been submitted to GenBank under accession numbers: Zebrafish STING (HE856619); EPC STING (HE856620); EPC IRF3 (HE856621); EPC IFN promoter (HE856618).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ARN Helicasas DEAD-box/metabolismo , Peces/metabolismo , Interferones/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Virus ADN/inmunología , Retículo Endoplásmico/metabolismo , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Peces/genética , Peces/inmunología , Expresión Génica , Regulación de la Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Unión Proteica , Transporte de Proteínas , Virus ARN/inmunología , Alineación de Secuencia , Virosis/genética , Virosis/inmunología , Virosis/virologíaRESUMEN
We report here the first full-length sequence of the eight ssRNA genome segments of the infectious salmon anemia virus (ISAV, Glesvaer/2/90 isolate), a salmonid orthomyxovirus-like. Comparison of ISAV genome sequence with those of others orthomyxovirus reveals low identity values, and a remarkable feature is the extremely long 5' end UTR of ISAV segments, which all contain an additional conserved motif of unknown function. In addition to the genome nucleotide sequence determination, specific mAbs have been produced through mice immunization with sucrose-purified ISAV. Four mAbs directed against the haemagglutinin-esterase glycoprotein, the nucleoprotein and free or actin-associated forms of the matrix protein have been characterized by (i) indirect fluorescent antibody test; (ii) virus neutralization; (iii) radioimmunoprecipitation and (iv) Western blot assays. These mAbs will potentially be useful for the development of new diagnostic tests, and the nucleotide sequences will help to establish a reverse genetics system for ISAV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Genoma Viral , Isavirus/genética , Isavirus/inmunología , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , ARN Viral/genética , Ensayo de Radioinmunoprecipitación , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-DeltaNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFP(max) reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFP(max) could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFP(max) and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins.
Asunto(s)
Virus de la Necrosis Hematopoyética Infecciosa/fisiología , Novirhabdovirus/fisiología , Infecciones por Rhabdoviridae/virología , Interferencia Viral/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Viral/genética , Genes Reporteros , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Virus de la Necrosis Hematopoyética Infecciosa/genética , Virus de la Necrosis Hematopoyética Infecciosa/patogenicidad , Datos de Secuencia Molecular , Mutación , Novirhabdovirus/genética , Novirhabdovirus/patogenicidad , Oncorhynchus mykiss , Plásmidos/genética , Recombinación Genética , Interferencia Viral/genética , Virulencia/genética , Virulencia/fisiología , Replicación ViralRESUMEN
Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ring-shaped nanoparticles (N(SRS)) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with N(SRS) either intramuscularly, or both intramuscularly and intranasally using Montanide ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non-vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the non-vaccinated control calves. Thus N(SRS) vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction.
Asunto(s)
Enfermedades de los Bovinos/prevención & control , Nucleoproteínas/inmunología , Infecciones por Virus Sincitial Respiratorio/veterinaria , Vacunas contra Virus Sincitial Respiratorio/inmunología , Proteínas Virales/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Bovinos , Enfermedades de los Bovinos/inmunología , Protección Cruzada , Inmunidad Celular , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Masculino , Datos de Secuencia Molecular , Nanopartículas , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Bovino/inmunología , Vacunas de Subunidad/inmunología , Carga ViralRESUMEN
The influenza A virus PB1-F2 protein, encoded by an alternative reading frame in the PB1 polymerase gene, displays a high sequence polymorphism and is reported to contribute to viral pathogenesis in a sequence-specific manner. To gain insights into the functions of PB1-F2, the molecular structure of several PB1-F2 variants produced in Escherichia coli was investigated in different environments. Circular dichroism spectroscopy shows that all variants have a random coil secondary structure in aqueous solution. When incubated in trifluoroethanol polar solvent, all PB1-F2 variants adopt an alpha-helix-rich structure, whereas incubated in acetonitrile, a solvent of medium polarity mimicking the membrane environment, they display beta-sheet secondary structures. Incubated with asolectin liposomes and SDS micelles, PB1-F2 variants also acquire a beta-sheet structure. Dynamic light scattering revealed that the presence of beta-sheets is correlated with an oligomerization/aggregation of PB1-F2. Electron microscopy showed that PB1-F2 forms amorphous aggregates in acetonitrile. In contrast, at low concentrations of SDS, PB1-F2 variants exhibited various abilities to form fibers that were evidenced as amyloid fibers in a thioflavin T assay. Using a recombinant virus and its PB1-F2 knock-out mutant, we show that PB1-F2 also forms amyloid structures in infected cells. Functional membrane permeabilization assays revealed that the PB1-F2 variants can perforate membranes at nanomolar concentrations but with activities found to be sequence-dependent and not obviously correlated with their differential ability to form amyloid fibers. All of these observations suggest that PB1-F2 could be involved in physiological processes through different pathways, permeabilization of cellular membranes, and amyloid fiber formation.
Asunto(s)
Amiloide/química , Membrana Celular/química , Virus de la Influenza A/química , Proteínas Virales/química , Acetonitrilos/química , Amiloide/genética , Amiloide/metabolismo , Amiloide/ultraestructura , Animales , Benzotiazoles , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Perros , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Mutación , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazoles/química , Trifluoroetanol/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.
Asunto(s)
Encéfalo/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/transmisión , Bazo/metabolismo , Animales , Encéfalo/patología , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , Células Cultivadas , Endopeptidasa K/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovinos , Bazo/patologíaRESUMEN
The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.
Asunto(s)
ARN Viral/metabolismo , Virus Sincitial Respiratorio Humano/química , Proteínas Virales/química , Proteínas Estructurales Virales/metabolismo , Sitios de Unión , Dicroismo Circular , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Virus Sincitial Respiratorio Humano/genética , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
A glycosyl-nucleoside-lipid self-assembles to give highly organized structures such as fibers and nanotubes, which can stabilize hydrogels; carbohydrate moieties provide a suitable environment to deliver nucleic acids into human cells.
Asunto(s)
Sistemas de Liberación de Medicamentos , Glucosa/química , Lípidos/química , Nanoestructuras/química , Oligonucleótidos/química , Portadores de Fármacos/química , Hepatocitos/química , Humanos , Hidrogeles/química , Sustancias Macromoleculares/química , Ensayo de Materiales , Estructura Molecular , Tamaño de la Partícula , Propiedades de Superficie , TransfecciónRESUMEN
BACKGROUND: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10-11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS). METHODOLOGY AND PRINCIPAL FINDINGS: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells. CONCLUSIONS/SIGNIFICANCE: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.
Asunto(s)
Nanopartículas , Virus Sincitiales Respiratorios/inmunología , Vacunas Virales/administración & dosificación , Administración Intranasal , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Vacunas Virales/inmunologíaRESUMEN
PrP(c) is the normal isoform of the prion protein which can be converted into PrP(Sc), the pathology-associated conformer in prion diseases. It contains two N-linked glycan chains attached to the C-proximal globular domain. While the biological functions of PrP(c) are still unknown, its ability to bind Cu(2+) is well documented. The main Cu(2+)-binding sites are located in the N-proximal, unstructured region of the molecule. Here we report that PrP(c) glycans influence the capacity of PrP(c) from sheep brain or cultured Rov cells to bind IMAC columns loaded with Cu(2+) or Co(2+). Using different anti-PrP antibodies and PrP(c) glycosylation mutants, we show that the full length non-glycosylated form of PrP(c) has a higher binding efficiency for column-bound Cu(2+) and Co(2+) than the corresponding glycosylated form. Our findings raise the possibility that the accessibility of the PrP(c) metal ion-binding sites might be controlled by the glycan chains.