RESUMEN
The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Oxidorreductasas de Alcohol/metabolismo , Núcleo Celular/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Antígenos Nucleares del Virus de Epstein-Barr/química , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/químicaRESUMEN
The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.
Asunto(s)
Virus del Sarampión/metabolismo , Virus del Sarampión/patogenicidad , Sarampión/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Antígenos CD/genética , Antígenos CD/metabolismo , Autofagia , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Gigantes/metabolismo , Células Gigantes/patología , Células Gigantes/virología , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Sarampión/genética , Sarampión/patología , Virus del Sarampión/genética , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
The Epstein-Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human ß-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the ß-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.
Asunto(s)
Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Núcleo Celular/virología , Citoplasma/metabolismo , Citoplasma/virología , Regulación hacia Abajo , Células HEK293 , Células HeLa , Humanos , Luciferasas de Renilla/genética , Mutación , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Empalme Serina-Arginina , Transactivadores/química , Transactivadores/genética , Técnicas del Sistema de Dos Híbridos , Globinas beta/genéticaRESUMEN
Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.
Asunto(s)
Autofagia/fisiología , Proteínas de Unión al GTP/metabolismo , Infecciones por Virus ARN/metabolismo , Infecciones por Virus ARN/transmisión , Virus ARN/metabolismo , Secuencia de Bases , Western Blotting , Biología Computacional , Proteínas de Unión al GTP/genética , Células HeLa , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Infecciones por Virus ARN/genética , Virus ARN/genética , ARN Interferente Pequeño , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/metabolismoRESUMEN
Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or "interologs". Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus-host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interaction assays: yeast two-hybrid (Y2H), and our recently described "high-throughput Gaussia princeps protein complementation assay" (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split G. princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus-host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism-host interactions.
Asunto(s)
Alphapapillomavirus/fisiología , Interacciones Huésped-Patógeno , Luciferasas/genética , Proteínas de Plantas/genética , Técnicas del Sistema de Dos Híbridos , Alphapapillomavirus/genética , Arecaceae/enzimología , Biomarcadores/metabolismo , Análisis por Conglomerados , Genotipo , Células HEK293 , Humanos , Luciferasas/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Filogenia , Proteínas de Plantas/biosíntesis , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Tropismo ViralRESUMEN
Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus-virus and virus-host lateral gene transfers. PhEVER (http://pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems.
Asunto(s)
Bases de Datos Genéticas , Evolución Molecular , Interacciones Huésped-Patógeno/genética , Virus/genética , Análisis por Conglomerados , Transferencia de Gen Horizontal , Genes Virales , Genoma Viral , Genómica , Filogenia , Homología de Secuencia , Interfaz Usuario-Computador , Proteínas Virales/química , Proteínas Virales/clasificación , Proteínas Virales/genética , Virus/clasificaciónRESUMEN
The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response.
Asunto(s)
Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Mapeo de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Humanos , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Replicación ViralRESUMEN
BACKGROUND: The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. RESULTS: We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. CONCLUSIONS: We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.
Asunto(s)
Interacciones Huésped-Patógeno , Mapeo de Interacción de Proteínas , Proteínas no Estructurales Virales/metabolismo , Flavivirus/patogenicidad , Infecciones por Flavivirus/virología , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Infectious diseases caused by viral agents kill millions of people every year. The improvement of prevention and treatment of viral infections and their associated diseases remains one of the main public health challenges. Towards this goal, deciphering virus-host molecular interactions opens new perspectives to understand the biology of infection and for the design of new antiviral strategies. Indeed, modelling of an infection network between viral and cellular proteins will provide a conceptual and analytic framework to efficiently formulate new biological hypothesis at the proteome scale and to rationalize drug discovery. Therefore, we present the first release of VirHostNet (Virus-Host Network), a public knowledge base specialized in the management and analysis of integrated virus-virus, virus-host and host-host interaction networks coupled to their functional annotations. VirHostNet integrates an extensive and original literature-curated dataset of virus-virus and virus-host interactions (2671 non-redundant interactions) representing more than 180 distinct viral species and one of the largest human interactome (10,672 proteins and 68,252 non-redundant interactions) reconstructed from publicly available data. The VirHostNet Web interface provides appropriate tools that allow efficient query and visualization of this infected cellular network. Public access to the VirHostNet knowledge-based system is available at http://pbildb1.univ-lyon1.fr/virhostnet.
Asunto(s)
Bases de Datos de Proteínas , Interacciones Huésped-Patógeno , Mapeo de Interacción de Proteínas , Proteínas Virales/metabolismo , Internet , Proteoma/metabolismo , Interfaz Usuario-Computador , Virosis/metabolismo , Virosis/virología , Fenómenos Fisiológicos de los VirusRESUMEN
Autophagy is a highly conserved, self-degradative pathway for clearance and recycling of cytoplasmic contents. This ubiquitous cell intrinsic process can be used as a defence mechanism against intracellular pathogens. Indeed autophagy is increased upon pathogen detection, and experimental extinction in vitro and in vivo of this cellular process has been demonstrated as a crucial role to control intracellular pathogens. Co-evolution between host-cells and pathogens has selected numerous micoorganisms able to avoid or usurp autophagy to their own benefit. Understanding mechanisms underlying the anti-microbial properties of autophagy as well as those used by certain pathogens to escape this cellular process might be crucial to manipulate this cellular function in order to prevent or treat infectious diseases.
Asunto(s)
Autofagia , Interacciones Huésped-Patógeno/fisiología , Animales , Fenómenos Fisiológicos Bacterianos , Células/microbiología , Células/parasitología , Células/virología , Células Eucariotas/fisiología , VIH/fisiología , Humanos , Interferón Tipo I/fisiología , Fusión de Membrana , Modelos Biológicos , Fagosomas/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Receptores de Reconocimiento de Patrones/fisiología , Selección Genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Infection caused by pathogens kills millions of people every year. Comprehensive understanding of molecular pathogen-host interactions, i.e. the infectome, is one of the key steps towards the development of novel diagnostic, therapeutic and preventive strategies. In this quest, progress in high-throughput << omics >> technologies applied to pathogens, i.e. infectomics, opens new perspectives toward systemic understanding of perturbations induced during infection. Deciphering the pathogen-host system also relies on the analytical and predictive power of molecular systems biology and by developing in silico models taking into account the whole picture of the molecules and their interactions. In this context, we have reconstructed a prototype of the human virtual infected cell based on 30 years of intensive research in the field of molecular virology. This model contains more than one hundred viral infectomes, including major human pathogens (HCV, HBV, HIV, HHV, HPV) and has led to the generation of novel systems-level hypotheses that could be suitable for the development of innovative antiviral strategies based on the control of cellular functions.
Asunto(s)
Antivirales/síntesis química , Antivirales/uso terapéutico , Interfaz Usuario-Computador , Virosis/genética , Genoma Viral , Humanos , Modelos Genéticos , Proteoma , Proteínas Virales/genética , Virosis/tratamiento farmacológico , Virus/genéticaRESUMEN
BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.
Asunto(s)
Células Dendríticas/fisiología , Macrófagos/fisiología , Microglía/fisiología , Osteoclastos/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Células Madre/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Proteínas Portadoras/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Osteoclastos/citología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Células Madre/citología , Células Madre/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Tirosina Quinasa 3 Similar a fmsRESUMEN
In addition to their essential role in adaptive immunity, dendritic cells (DCs) participate in innate immunity. In the context of measles virus (MV) or cytomegalovirus infections, they develop cytotoxic functions that may contribute in vivo to the elimination of virus-infected cells, but that also kill infected and noninfected T lymphocytes. Because the human immunodeficiency virus (HIV) induces T cell depletion through mechanisms that are still obscure, we investigated its ability to trigger DC cytotoxicity. When incubated with HIV, monocyte-derived DCs induced apoptosis in MDA-231 cells, which are sensitive to MV-induced DC cytotoxicity, and in uninfected as well as HIV-infected H9 CD4+ T cell lines. This apoptosis was inhibited by a mixture of FasL, TRAIL, TNF-alpha, and TWEAK inhibitors. Indeed, HIV infection induced or enhanced sensitivity to TRAIL, TNF-alpha, and TWEAK in H9 cells. Moreover, dendritic cells incubated with HIV-1 BAL or a wildtype HIV-1 isolate induced apoptosis in autologous primary CD4+ T lymphocytes, infected or not with a wild-type HIV-1 isolate. Therefore, induction of DC cytotoxicity by HIV may be relevant to in vivo HIV infection. Induction of cytotoxicity in DCs by HIV might contribute to HIV-associated T cell depletion through induction of apoptosis, especially in the early stages of infection. It may also contribute to elimination of infected cells in vivo, thereby enhancing cross-presentation of HIV by DCs. Therefore this new cytotoxic function of DCs may play an important role in innate and adaptive immunity during HIV infection.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/virología , VIH-1/patogenicidad , Proteínas Reguladoras de la Apoptosis , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Células Cultivadas , Citotoxicidad Inmunológica , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , Humanos , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-γ-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-γ. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(low)HLADR(low) CD1a(+) MCL1(+), did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high) HLADR(low)) pre-M2 (CCL22(+) CD206(+) CD163(+) IL1RN(+) IL-10(-) CXCL10(-) IL-12(-)) mixed (CD1a(+) CD14+ CD68(+)) macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-κB, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic cells, blocking IL-17A, may be either dangerous or beneficial for cancer outcomes, thus contributing to the apparent controversy around the role of IL-17A in cancer.
Asunto(s)
Antineoplásicos/farmacología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Resistencia a Antineoplásicos , Interleucina-17/farmacología , Monocitos/citología , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/metabolismo , Resistencia a Antineoplásicos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunofenotipificación , Interferón gamma/farmacología , Antígenos de Histocompatibilidad Menor , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , FN-kappa B/metabolismo , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transcripción GenéticaRESUMEN
Using global approaches and high-throughput technologies in virology brings a new vision of the infections physiology and allows the identification of cellular factors, mandatory for viral life cycle, that could be targeted by original therapeutic agents. It opens perspectives for the treatment of viral infections by acting on cellular pathways that the virus must use for its own replication. Combining these new molecules with classical antiviral drugs and immunomodulators diversifies and enlarges the antiviral arsenal and contributes to fight drug resistance. Our laboratory and others are constructing virus-human interactomes to propose a comprehensive analysis of viral infection at the cellular level. Studying these infection maps, where the viral infection can be visualized as perturbation of the human protein-protein interaction network, and identifying the biological functions that are impaired by these perturbations may lead to discovery of new therapeutic targets. These virus-human interaction maps are constructed in a stringent yeast two-hybrid system by screening human cDNA libraries with viral proteins as bait and integrating interactions mined from literature and public databases.
Asunto(s)
Interacciones Huésped-Patógeno , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/metabolismo , Fenómenos Fisiológicos de los Virus , Virus/metabolismo , ADN Viral/genética , ADN Viral/aislamiento & purificación , Bases de Datos de Proteínas , Biblioteca de Genes , Humanos , Sistemas de Lectura Abierta/genética , Transformación Genética , Proteínas Virales/genética , Virus/genética , Levaduras/citología , Levaduras/genéticaRESUMEN
Several intracellular pathogens have the ability to avoid or exploit the otherwise destructive process of autophagy. RNA viruses are constantly confronted with cellular autophagy, and several of them hijack autophagy during the infectious cycle to improve their own replication. Nevertheless, our knowledge of viral molecular strategies used to manipulate autophagy remains limited. Our study allowed the identification of molecular interactions between 44 autophagy-associated proteins and 83 viral proteins belonging to five different RNA virus families. This interactome revealed that the autophagy network machinery is highly targeted by RNA viruses. Interestingly, whereas some autophagy-associated proteins are targeted by only one RNA virus family, others are recurrent targets of several families. Among them, we found IRGM as the most targeted autophagy-associated protein. Downregulation of IRGM expression prevents autophagy induction by measles virus, HCV and HIV-1, and compromises viral replication. Our work combined interactomic and analytical approaches to identify potential pathogen virulence factors targeting autophagy.
RESUMEN
Entry into a cell submits viruses to detection by pattern recognition receptors (PRRs) leading to an early innate anti-viral response. Several viruses evolved strategies to avoid or subvert PRR recognition at the step of virus entry to promote infection. Whereas viruses mostly escape from soluble PRR detection, endocytic/phagocytic PRRs, such as the mannose receptor or DC-SIGN, are commonly used for virus entry. Moreover, virion-incorporated proteins may also offer viruses a way to dampen anti-viral innate immunity upon virus entry, and entering viruses might usurp autophagy to improve their own infectivity.
Asunto(s)
Inmunidad Innata , Virosis/inmunología , Virosis/virología , Internalización del Virus , Animales , Humanos , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Receptores Virales/genética , Receptores Virales/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Virus/genética , Virus/inmunologíaRESUMEN
Autophagy is a degradative mechanism involved in cell protection against invading pathogens. Although the autophagic process is well characterized, the molecular pathways leading to its activation upon pathogen binding remain poorly understood. Our recent work demonstrates that the cell surface pathogen receptor CD46 induces autophagy upon pathogen recognition. The molecular pathway linking CD46 to the autophagosome machinery relies on the scaffold protein GOPC and on the autophagosome formation complex Beclin 1/VPS34. The CD46-dependent autophagy is critical to an early control of infection.
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Autofagia/inmunología , Proteína Cofactora de Membrana/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Interacciones Huésped-Patógeno , Ratones , Fagosomas/metabolismoRESUMEN
While the antiviral response during measles virus (MeV) infection is documented, the contribution of the hosting cell type to the type I interferon (IFN-alpha/beta) response is still not clearly established. Here, we report that a signature heterogeneity of the IFN-alpha/beta response according to the cell type. The MeV tropism dictated by the expression of appropriate cellular receptor appeared to be crucial for epithelial cells. For conventional DCs (cDCs), the maturation state played a prominent role. In response to both wild type MeV isolates and laboratory/vaccine strains, immature cDCs produced higher levels of IFN-alpha than mature cDCs, despite the reduced expression levels of both CD46 and CD150 receptors by the former ones. While in epithelial cells and cDCs the MeV transcription was required to activate the IFN-alpha/beta response, plasmacytoid DCs (pDCs) rapidly produced large amounts of IFN-alpha mostly independently of the viral infection cycle. This argues for a significant contribution of pDCs in response to MeV infection and/or vaccination.
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Diferenciación Celular , Células Dendríticas/inmunología , Endocitosis , Células Epiteliales/inmunología , Interferón Tipo I/inmunología , Virus del Sarampión/fisiología , Sarampión/inmunología , Receptores Virales/inmunología , Antígenos CD/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/virología , Células Epiteliales/citología , Células Epiteliales/virología , Humanos , Sarampión/fisiopatología , Sarampión/virología , Virus del Sarampión/genética , Virus del Sarampión/inmunología , Proteína Cofactora de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Tropismo ViralRESUMEN
BACKGROUND: High-throughput screening of protein-protein interactions opens new systems biology perspectives for the comprehensive understanding of cell physiology in normal and pathological conditions. In this context, yeast two-hybrid system appears as a promising approach to efficiently reconstruct protein interaction networks at the proteome-wide scale. This protein interaction screening method generates a large amount of raw sequence data, i.e. the ISTs (Interaction Sequence Tags), which urgently need appropriate tools for their systematic and standardised analysis. FINDINGS: We develop pISTil, a bioinformatics pipeline combined with a user-friendly web-interface: (i) to establish a standardised system to analyse and to annotate ISTs generated by two-hybrid technologies with high performance and flexibility and (ii) to provide high-quality protein-protein interaction datasets for systems-level approach. This pipeline has been validated on a large dataset comprising more than 11.000 ISTs. As a case study, a detailed analysis of ISTs obtained from yeast two-hybrid screens of Hepatitis C Virus proteins against human cDNA libraries is also provided. CONCLUSION: We have developed pISTil, an open source pipeline made of a collection of several applications governed by a Perl script. The pISTil pipeline is intended to laboratories, with IT-expertise in system administration, scripting and database management, willing to automatically process large amount of ISTs data for accurate reconstruction of protein interaction networks in a systems biology perspective. pISTil is publicly available for download at http://sourceforge.net/projects/pistil.