RESUMEN
Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.
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Interferones , Mitocondrias , Ratones , Animales , Mitocondrias/metabolismo , Virus del Sarampión , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , ADN MitocondrialRESUMEN
Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M > ORF3a > N>ORF6 > ORF7a > ORF8 > S > E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.
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Biología Computacional/métodos , Genoma Viral , ARN Viral/genética , SARS-CoV-2/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura AbiertaRESUMEN
Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Muerte Celular , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Mapas de Interacción de Proteínas , Proteómica , Proteínas Represoras/genética , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.
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Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Animales , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/metabolismo , Ebolavirus/fisiología , Antivirales/farmacología , Antivirales/uso terapéutico , MamíferosRESUMEN
COVID-19 can lead to acute respiratory syndrome, which can be due to dysregulated immune signaling. We analyze the distribution of CpG dinucleotides, a pathogen-associated molecular pattern, in the SARS-CoV-2 genome. We characterize CpG content by a CpG force that accounts for statistical constraints acting on the genome at the nucleotidic and amino acid levels. The CpG force, as the CpG content, is overall low compared with other pathogenic betacoronaviruses; however, it widely fluctuates along the genome, with a particularly low value, comparable with the circulating seasonal HKU1, in the spike coding region and a greater value, comparable with SARS and MERS, in the highly expressed nucleocapside coding region (N ORF), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA. This dual nature of CpG content could confer to SARS-CoV-2 the ability to avoid triggering pattern recognition receptors upon entry, while eliciting a stronger response during replication. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic, finding a signature of CpG loss in regions with a greater CpG force. Sequence motifs preceding the CpG-loss-associated loci in the N ORF match recently identified binding patterns of the zinc finger antiviral protein. Using a model of the viral gene evolution under human host pressure, we find that synonymous mutations seem driven in the SARS-CoV-2 genome, and particularly in the N ORF, by the viral codon bias, the transition-transversion bias, and the pressure to lower CpG content.
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COVID-19/genética , Islas de CpG , Evolución Molecular , Genoma Viral , ARN Viral/genética , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidadRESUMEN
Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed DI-tector, a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. DI-tector confirmed the presence of 5' cb genomes in cells infected with measles virus (MV). DI-tector also identified a novel 5' cb genome, as well as a variety of 3' cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of DI-tector to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of DI-tector. We propose DI-tector as a universal tool for the unbiased detection of DI viral genomes, including 5' cb/sb DI genomes, in NGS data.
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Virus Defectuosos/genética , Genoma Viral , Genómica , Programas Informáticos , Línea Celular , Biología Computacional/métodos , Genes de ARNr , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Viral , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Replicación ViralRESUMEN
Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5' copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5' copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six." We correlated the presence of DI-RNAs with a larger stimulation of the IFN-ß pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling.IMPORTANCE Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.
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Genoma Viral , Helicasa Inducida por Interferón IFIH1/metabolismo , Virus del Sarampión/genética , ARN Helicasas/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Receptores de Ácido Retinoico/metabolismo , Línea Celular , Humanos , Inmunidad Innata , Interferón beta/metabolismo , Sarampión/virología , Vacuna Antisarampión/genética , Vacuna Antisarampión/inmunología , Virus del Sarampión/patogenicidad , Nucleocápside/metabolismo , ARN Viral/inmunología , Transducción de SeñalRESUMEN
The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.
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Interacciones Huésped-Patógeno , Virus del Sarampión , Genética Inversa , Proteínas Virales , Virus del Sarampión/genética , Humanos , Interacciones Huésped-Patógeno/genética , Genética Inversa/métodos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Técnicas del Sistema de Dos Híbridos , Replicación Viral , Espectrometría de Masas , Mapeo de Interacción de Proteínas/métodos , Sarampión/virología , Sarampión/metabolismo , Animales , Unión ProteicaRESUMEN
Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.
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Proteína 58 DEAD Box , Virus de la Rabia , Humanos , Línea Celular , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Rabia/inmunología , Rabia/virología , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Virus de la Rabia/genética , Virus de la Rabia/patogenicidad , Receptores Inmunológicos/metabolismo , ARN Viral/genética , Transducción de Señal , Replicación ViralRESUMEN
RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5' copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.
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Virus del Sarampión/metabolismo , Nucleoproteínas/metabolismo , ARN Viral/genética , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Virus del Sarampión/genética , Proteínas de la Nucleocápside , Nucleoproteínas/aislamiento & purificación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Receptores Inmunológicos , Proteínas Recombinantes/metabolismo , Células Vero , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.
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Interacciones Huésped-Patógeno , Virus del Sarampión/fisiología , Sarampión/virología , Animales , Chlorocebus aethiops , ARN Helicasas DEAD-box/aislamiento & purificación , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1 , Sarampión/metabolismo , Virus del Sarampión/genética , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Organismos Modificados Genéticamente , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Genética Inversa , Factor de Transcripción STAT1/aislamiento & purificación , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/aislamiento & purificación , Factor de Transcripción STAT2/metabolismo , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem , Células Vero , Factores de Virulencia/genética , Factores de Virulencia/aislamiento & purificación , Factores de Virulencia/metabolismo , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. SARS-CoV-2 is characterized by an important capacity to circumvent the innate immune response. The early interferon (IFN) response is necessary to establish a robust antiviral state. However, this response is weak and delayed in COVID-19 patients, along with massive pro-inflammatory cytokine production. This dysregulated innate immune response contributes to pathogenicity and in some individuals leads to a critical state. Characterizing the interplay between viral factors and host innate immunity is crucial to better understand how to manage the disease. Moreover, the constant emergence of new SARS-CoV-2 variants challenges the efficacy of existing vaccines. Thus, to control this virus and readjust the antiviral therapy currently used to treat COVID-19, studies should constantly be re-evaluated to further decipher the mechanisms leading to SARS-CoV-2 pathogenesis. Regarding the role of the IFN response in SARS-CoV-2 infection, in this review we summarize the mechanisms by which SARS-CoV-2 evades innate immune recognition. More specifically, we explain how this virus inhibits IFN signaling pathways (IFN-I/IFN-III) and controls interferon-stimulated gene (ISG) expression. We also discuss the development and use of IFNs and potential drugs controlling the innate immune response to SARS-CoV-2, helping to clear the infection.
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Tratamiento Farmacológico de COVID-19 , Interferón Tipo I , Antivirales/farmacología , Antivirales/uso terapéutico , Humanos , Evasión Inmune , Inmunidad Innata , Interferones/uso terapéutico , Pandemias , SARS-CoV-2RESUMEN
Pattern recognition receptors (PRRs) protect against microbial invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specifically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphosphate extremity. Further, we found that HIV-1 triggered a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a transcriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endogenous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.
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A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.
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Factor de Crecimiento Epidérmico/metabolismo , Interferón Tipo I/metabolismo , Virus de la Parainfluenza 3 Humana/metabolismo , Factores de Virulencia/metabolismo , Animales , Sitios de Unión , Recuento de Células , Línea Celular , Chlorocebus aethiops , Factor 4E Eucariótico de Iniciación/metabolismo , Citometría de Flujo , Proteína Adaptadora GRB2/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Virus de la Parainfluenza 3 Humana/patogenicidad , Fosforilación , Mapeo de Interacción de Proteínas , Reproducibilidad de los Resultados , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Células Vero , Proteínas Virales/metabolismoRESUMEN
COVID-19 can lead to acute respiratory syndrome, which can be due to dysregulated immune signaling. We analyze the distribution of CpG dinucleotides, a pathogen-associated molecular pattern, in the SARS-CoV-2 genome. We find that the CpG content, which we characterize by a force parameter that accounts for statistical constraints acting on the genome at the nucleotidic and amino-acid levels, is, on average, low compared to other pathogenic betacoronaviruses. However, the CpG force widely fluctuates along the genome, with a particularly low value, comparable to the circulating seasonal HKU1, in the spike coding region and a greater value, comparable to SARS and MERS, in the highly expressed nucleocapside coding region (N ORF), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA. This dual nature of CpG content could confer to SARS-CoV-2 the ability to avoid triggering pattern recognition receptors upon entry, while eliciting a stronger response during replication. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic, finding a signature of CpG loss in regions with a greater CpG force. Sequence motifs preceding the CpG-loss-associated loci in the N ORF match recently identified binding patterns of the Zinc finger Anti-viral Protein. Using a model of the viral gene evolution under human host pressure, we find that synonymous mutations seem driven in the SARS-CoV-2 genome, and particularly in the N ORF, by the viral codon bias, the transition-transversion bias and the pressure to lower CpG content.
RESUMEN
SARS-CoV-2 infection can lead to acute respiratory syndrome in patients, which can be due in part to dysregulated immune signalling. We analyze here the occurrences of CpG dinucleotides, which are putative pathogen-associated molecular patterns, along the viral sequence. Carrying out a comparative analysis with other ssRNA viruses and within the Coronaviridae family, we find the CpG content of SARS-CoV-2, while low compared to other betacoronaviruses, widely fluctuates along its primary sequence. While the CpG relative abundance and its associated CpG force parameter are low for the spike protein (S) and comparable to circulating seasonal coronaviruses such as HKU1, they are much greater and comparable to SARS and MERS for the 3'-end of the viral genome. In particular, the nucleocapsid protein (N), whose transcripts are relatively abundant in the cytoplasm of infected cells and present in the 3'UTRs of all subgenomic RNA, has high CpG content. We speculate this dual nature of CpG content can confer to SARS-CoV-2 high ability to both enter the host and trigger pattern recognition receptors (PRRs) in different contexts. We then investigate the evolution of synonymous mutations since the outbreak of the COVID-19 pandemic. Using a new application of selective forces on dinucleotides to estimate context driven mutational processes, we find that synonymous mutations seem driven both by the viral codon bias and by the high value of the CpG force in the N protein, leading to a loss in CpG content. Sequence motifs preceding these CpG-loss-associated loci match recently identified binding patterns of the Zinc Finger anti-viral Protein (ZAP) protein. Funding: This work was partially supported by the ANR19 Decrypted CE30-0021-01 grants. B.G. was supported by National Institutes of Health grants 7R01AI081848-04, 1R01CA240924-01, a Stand Up to Cancer - Lustgarten Foundation Convergence Dream Team Grant, and The Pershing Square Sohn Prize - Mark Foundation Fellow supported by funding from The Mark Foundation for Cancer Research.
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Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.
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Factor 3 de Iniciación Eucariótica/metabolismo , Biosíntesis de Proteínas , Virus de la Rabia/patogenicidad , Proteínas de la Matriz Viral/metabolismo , Secuencia de Aminoácidos , Factor 3 de Iniciación Eucariótica/inmunología , Inmunoprecipitación , Datos de Secuencia Molecular , Mutación , Subunidades de Proteína/metabolismo , ARN Mensajero/química , ARN Viral/química , Ribosomas/virología , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos Híbridos , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.
Asunto(s)
Antivirales/metabolismo , Proteína 58 DEAD Box/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Chlorocebus aethiops , Proteína 58 DEAD Box/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiología , Células HEK293 , Células HeLa , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Mengovirus/genética , Mengovirus/fisiología , Unión Proteica , ARN Helicasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Receptores Inmunológicos , Transducción de Señal/genética , Células VeroRESUMEN
RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-ß production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as "social RNAs" to trigger a host response during infection.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proteína 58 DEAD Box/inmunología , Células HEK293 , Interacciones Microbiota-Huesped , Humanos , Inmunidad Innata , Interferón beta/metabolismo , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Ratones , ARN Bacteriano/metabolismo , Transducción de Señal/inmunología , Virulencia/inmunologíaRESUMEN
The flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. In vitro production of fragments of the DENV genome confirmed the NGS data and revealed that the 5' end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells.