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Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.
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The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.
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Antivirales , ADN Helicasas , Antivirales/metabolismo , ADN Helicasas/metabolismo , Interferones/metabolismo , Nucleósidos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.
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COVID-19 , SARS-CoV-2 , Humanos , Acoplamiento Viral , Ensayos Analíticos de Alto Rendimiento , Unión ProteicaRESUMEN
Antibodies are natural antivirals generated by the vertebrate immune system in response to viral infection or vaccination. Unsurprisingly, they are also key molecules in the virologist's molecular toolbox. With new developments in methods for protein engineering, protein functionalization and application, smaller antibody-derived fragments are moving in focus. Among these, camelid-derived nanobodies play a prominent role. Nanobodies can replace full-sized antibodies in most applications and enable new possible applications for which conventional antibodies are challenging to use. Here we review the versatile nature of nanobodies, discuss their promise as antiviral therapeutics, for diagnostics, and their suitability as research tools to uncover novel aspects of viral infection and disease.
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Anticuerpos de Dominio Único , Virus , Anticuerpos , Proteínas , Anticuerpos de Dominio Único/metabolismoRESUMEN
We present a comprehensive overview of the dependency of several Old World alphaviruses for the host protein G3BP. Based on their replication ability in G3BP-deleted cells, Old World alphaviruses can be categorized into two groups, being either resistant or sensitive to G3BP deletion. We observed that all sensitive viruses have an Arg residue at the P4 position of the cleavage site between the nonstructural protein P1 (nsP1) and nsP2 regions of the replicase precursor polyprotein (1/2 site), while a different residue is found at this site in viruses resistant to G3BP deletion. Swapping this residue between resistant and sensitive viruses also switches the G3BP deletion sensitivity. In the absence of G3BP, chikungunya virus (CHIKV) replication is at the limit of detection. The P4 Arg-to-His substitution partially rescues this defect. The P4 residue of the 1/2 site is known to play a regulatory role during processing at this site, and we found that if processing is blocked, the influence of the P4 residue on the sensitivity to G3BP deletion is abolished. Immunofluorescence experiments with CHIKV replicase with manipulated processing indicate that the synthesis of double-stranded RNA is defective in the absence of G3BP and suggest a role of G3BP during negative-strand RNA synthesis. This study provides a functional link between the host protein G3BP and the P4 residue of the 1/2 site for viral RNA replication of Old World alphaviruses. While this suggests a link between G3BP proteins and viral replicase polyprotein processing, we propose that G3BP proteins do not have a regulatory role during polyprotein processing.IMPORTANCE Old World alphaviruses comprise several medically relevant viruses, including chikungunya virus and Ross River virus. Recurrent outbreaks and the lack of antivirals and vaccines demand ongoing research to fight the emergence of these infectious diseases. In this context, a thorough investigation of virus-host interactions is critical. Here, we highlight the importance of the host protein G3BP for several Old World alphaviruses. Our data strongly suggest that G3BP plays a crucial role for the activity of the viral replicase and, thus, the amplification of the viral RNA genome. To our knowledge, the present work is the first to provide a functional link between the regulation of viral polyprotein processing and RNA replication and a host factor for alphaviruses. Moreover, the results of this study raise several questions about the fundamental regulatory mechanisms that dictate the activity of the viral replicase, thereby paving the way for future studies.
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Virus Chikungunya/fisiología , ADN Helicasas/genética , Eliminación de Gen , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Aedes , Animales , Antivirales/farmacología , Línea Celular Tumoral , Fiebre Chikungunya/virología , Cricetinae , Genoma Viral , Humanos , Poliproteínas/metabolismo , Unión Proteica , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs.
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Proteínas Portadoras/metabolismo , Fiebre Chikungunya/metabolismo , Virus Chikungunya/fisiología , ADN Helicasas/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Virus de los Bosques Semliki/fisiología , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Fiebre Chikungunya/genética , Fiebre Chikungunya/patología , Cricetinae , ADN Helicasas/genética , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Dominios Proteicos , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
Stress granules (SGs) are dynamic RNA-protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.
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Arabidopsis/metabolismo , Gránulos Citoplasmáticos/metabolismo , Estrés Fisiológico , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Fenotipo , Filogenia , Unión Proteica , Dominios ProteicosRESUMEN
Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.
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Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/virología , Alphavirus/fisiología , Glucosa/metabolismo , Interacciones Huésped-Patógeno , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alphavirus/patogenicidad , Animales , Células Cultivadas , Cricetinae , Activación Enzimática , Glucólisis/fisiología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Virus del Río Ross/fisiología , Virus de los Bosques Semliki/fisiología , VirulenciaRESUMEN
RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3-4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.
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Infecciones por Alphavirus/genética , Infecciones por Alphavirus/virología , Arenavirus del Viejo Mundo/fisiología , ARN Mensajero/genética , Infecciones por Alphavirus/metabolismo , Animales , Arenavirus del Viejo Mundo/genética , Línea Celular , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Ratones , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Replicación ViralRESUMEN
With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.
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Mutación Puntual , Alelos , Animales , Femenino , Genes Letales , Ligamiento Genético , Masculino , Ratones , Linaje , Fenotipo , Sitios de Carácter CuantitativoRESUMEN
Acute respiratory virus infections predispose the cystic fibrosis (CF) lung to chronic bacterial colonization, which contributes to high mortality. For reasons unknown, respiratory virus infections have a prolonged duration in CF. Here, we demonstrate that mice carrying the most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutation in humans, ΔF508, show increased morbidity and mortality following infection with a common human enterovirus. ΔF508 mice demonstrated impaired viral clearance, a slower type I interferon response and delayed production of virus-neutralizing antibodies. While the ΔF508 mice had a normal immune cell repertoire, unchanged serum immunoglobulin concentrations and an intact immune response to a T-cell-independent antigen, their response to a T-cell-dependent antigen was significantly delayed. Our studies reveal a novel function for CFTR in antiviral immunity and demonstrate that the ΔF508 mutation in cftr is coupled to an impaired adaptive immune response. This important insight could open up new approaches for patient care and treatment.
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Inmunidad Adaptativa/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/inmunología , Inmunidad Innata/genética , Mutación , Virosis/etiología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Codón , Fibrosis Quística/complicaciones , Modelos Animales de Enfermedad , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Interferón-alfa/biosíntesis , Ratones , Poli I-C/inmunología , Tasa de Supervivencia , Carga ViralRESUMEN
Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE: Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections.
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Alcaloides/farmacología , Antivirales/farmacología , Berberina/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Fiebre Chikungunya/metabolismo , Cricetinae , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Células Vero , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.
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Infecciones por Alphavirus/virología , Virus del Río Ross/genética , Virus del Río Ross/inmunología , Proteínas Estructurales Virales/genética , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/fisiopatología , Animales , Línea Celular , Línea Celular Tumoral , Virus Chikungunya/inmunología , Chlorocebus aethiops , Cricetinae , Humanos , Concentración de Iones de Hidrógeno , Ratones , Mutación , Sistemas de Lectura , Virus del Río Ross/patogenicidad , Eliminación de Secuencia , Células Vero , Carga Viral , Proteínas Estructurales Virales/análisis , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunología , Replicación ViralRESUMEN
The Ras-GAP SH3 domain-binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.
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Proteínas Portadoras , Gránulos Citoplasmáticos/química , Proteínas de Unión al ADN , Herpesvirus Humano 1 , Modelos Moleculares , Proteínas Virales , Secuencias de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The proinflammatory microenvironment in the respiratory airway induces maturation of both resident and infiltrating dendritic cells (DCs) upon influenza A virus (IAV) infection. This results in upregulation of antiviral pathways as well as modulation of endocytic processes, which affect the susceptibility of DCs to IAV infection. Therefore, it is highly relevant to understand how IAV interacts with and infects mature DCs. To investigate how different subsets of human myeloid DCs (MDCs) involved in tissue inflammation are affected by inflammatory stimulation during IAV infection, we stimulated primary blood MDCs and inflammatory monocyte-derived DCs (MDDCs) with TLR ligands, resulting in maturation. Interestingly, MDDCs but not MDCs were protected against IAV infection after LPS (TLR4) stimulation. In contrast, stimulation with TLR7/8 ligand protected MDCs but not MDDCs from IAV infection. The reduced susceptibility to IAV infection correlated with induction of type I IFNs. We found that differential expression of TLR4, TRIF, and MyD88 in the two MDC subsets regulated the ability of the cells to enter an antiviral state upon maturation. This difference was functionally confirmed using small interfering RNA and inhibitors. Our data show that different human MDC subsets may play distinct roles during IAV infection, as their capacity to induce type I IFNs is dependent on TLR-specific maturation, resulting in differential susceptibility to IAV infection.
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Células Dendríticas/metabolismo , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/metabolismo , Células Mieloides/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/virología , Técnicas de Silenciamiento del Gen , Humanos , Gripe Humana/genética , Interferón Tipo I/biosíntesis , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/virología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Poli I-C/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/genéticaRESUMEN
UNLABELLED: Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE: SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human pathogen, causing high fever and joint pain, while SFV is a low-pathogenic model virus, albeit neuropathogenic in mice. We show that both SFV and CHIKV activate the prosurvival PI3K-Akt-mTOR pathway in cells but greatly differ in their capacities to do so: Akt is strongly and persistently activated by SFV infection but only moderately activated by CHIKV. We mapped this activation capacity to a region in nonstructural protein 3 (nsP3) of SFV and could functionally transfer this region to CHIKV. Akt activation is linked to the subcellular dynamics of replication complexes, which are efficiently internalized from the cell periphery for SFV but not CHIKV. This difference in signal pathway stimulation and replication complex localization may have implications for pathology.
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Virus Chikungunya/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/genética , Virus de los Bosques Semliki/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Infecciones por Alphavirus/virología , Androstadienos/farmacología , Animales , Línea Celular Tumoral , Virus Chikungunya/genética , Cricetinae , Activación Enzimática , Humanos , Ratones , Naftiridinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Estructura Terciaria de Proteína/genética , Virus de los Bosques Semliki/genética , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral , WortmaninaRESUMEN
Stress granules are induced in many different viral infections, and in turn are inhibited by the expression of viral proteins or RNAs. It is therefore evident that these bodies are not compatible with efficient viral replication, but the mechanism by which they act to restrict viral gene expression or genome replication is not yet understood. This article discusses a number of methods that can be employed to gain a more complete understanding of the relationship between cellular SGs and viral RNA and protein synthesis in cells infected with diverse viruses.
Asunto(s)
Proteínas Virales/análisis , Virología/métodos , Interacciones Huésped-Patógeno , Microscopía/métodos , ARN Viral/análisis , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Estrés FisiológicoRESUMEN
The Old World alphaviruses block stress granule assembly by sequestration of RasGAP SH3-domain binding protein (G3BP). Here, we show that the proline-rich sequences in the hypervariable domain of nonstructural protein 3 (nsP3) of both Semliki Forest virus and Chikungunya virus were dispensable for binding to G3BP. nsP3 variants with or without this domain colocalized with G3BP. Furthermore, we show that the C-terminal repeat motifs of nsP3 were sufficient for G3BP binding.
Asunto(s)
Virus Chikungunya/fisiología , Interacciones Huésped-Patógeno , Virus de los Bosques Semliki/fisiología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , ADN Helicasas/inmunología , Proteínas de Homeodominio/inmunología , Inmunidad Humoral/fisiología , Factores de Transcripción de Tipo Kruppel/inmunología , Proteínas/inmunología , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Células Cultivadas , ADN Helicasas/genética , Proteínas de Homeodominio/genética , Inmunidad Innata/fisiología , Péptidos y Proteínas de Señalización Intracelular , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Noqueados , Mutación , Proteínas/genética , Linfocitos T/inmunología , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Secondary infections with Streptococcus pneumoniae (SP) are frequently observed following influenza A virus (IAV) infection and have a substantial impact on global health. Despite this, the basis for the disease progression is incompletely understood. To investigate the effect of co-infection on human monocyte-derived dendritic cells (MDDCs) we analysed the expression of clinically important pro-inflammatory and immune-modulatory cytokines. IAV infection or treatment with supernatants from IAV-infected cell cultures resulted in priming of the DCs which subsequently influenced the production of IL-12p70, as well as IL-6, following SP infection. Co-infection of the same cell was not required but this effect was dependent on the time, dose and duration of the infections, as well as pathogen viability, bacterial uptake and endosome acidification. Bacterially infected cells were characterized as the main producers of IL-12p70. Finally, we showed that type I interferons were primarily responsible for the priming of IL-12p70 that was observed by infection with IAV. These results provide a probable mechanism for the elevated levels of particular cytokines observed in IAV and SP co-infected cell cultures with implications for the pathogenic outcome observed during in vivo infection.