RESUMEN
Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.
Asunto(s)
Inmunidad Innata , Interferones/fisiología , Mapeo de Interacción de Proteínas , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Expresión Génica , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/genética , Espectrometría de Masas , Receptores CCR4/metabolismo , Receptores de Péptidos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismoRESUMEN
Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.
Asunto(s)
Virus de la Influenza A , Virus de la Influenza A/genética , Replicación Viral/fisiología , Transcripción Genética , Nucleotidiltransferasas/metabolismo , Genómica , Glucólisis , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Interferón-alfa/farmacología , SARS-CoV-2/efectos de los fármacos , Transcriptoma , Replicación Viral/efectos de los fármacos , Animales , COVID-19/inmunología , COVID-19/virología , Chlorocebus aethiops , Clonación Molecular , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Ratones , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/farmacología , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Transducción de Señal , Células VeroRESUMEN
Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.
Asunto(s)
COVID-19/metabolismo , Furina/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adaptación Fisiológica , Animales , COVID-19/genética , COVID-19/virología , Chlorocebus aethiops , Efecto Citopatogénico Viral , Furina/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , SARS-CoV-2/genética , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Replicación ViralRESUMEN
Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Fenamatos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , SARS-CoV-2/efectos de los fármacos , Células A549 , Adulto , Animales , COVID-19/metabolismo , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/metabolismo , SARS-CoV-2/fisiología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.
Asunto(s)
Virus de la Influenza A/metabolismo , Gripe Humana/virología , Ribonucleoproteínas/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/metabolismo , Sistema de Señalización de MAP Quinasas , Señales de Exportación Nuclear , Ribonucleoproteínas/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.
Asunto(s)
COVID-19 , Gripe Humana , Adulto , Humanos , Enzima Convertidora de Angiotensina 2 , Pulmón/patología , Macrófagos Alveolares/metabolismo , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Tropismo ViralRESUMEN
Influenza viruses are small RNA viruses with a genome of about 13 kb. Because of this limited coding capacity, viral proteins have evolved to fulfil multiple functions in the infected cell. This implies that there must be mechanisms allowing to dynamically direct protein action to a distinct activity in a spatio-temporal manner. Furthermore, viruses exploit many cellular processes, which also have to be dynamically regulated during the viral replication cycle. Phosphorylation and dephosphorylation of proteins are fundamental for the control of many cellular responses. There is accumulating evidence that this mechanism represents a so far underestimated level of regulation in influenza virus replication. Here, we focus on the current knowledge of dynamics of phospho-modifications in influenza virus replication and show recent examples of findings underlining the crucial role of phosphorylation in viral transport processes as well as activation and counteraction of the innate immune response.
Asunto(s)
Gripe Humana , Humanos , Inmunidad Innata , Replicación ViralRESUMEN
The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN-antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN-suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN-antagonistic properties. Mutation of this position leads to enhanced activation of the IFN-mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.
Asunto(s)
Virus de la Influenza B , Interferón Tipo I/antagonistas & inhibidores , Proteínas de la Nucleocápside/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Proteínas Virales/inmunología , Células A549 , Animales , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Virus de la Influenza B/enzimología , Virus de la Influenza B/inmunología , Gripe Humana/virología , Células VeroRESUMEN
Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hübel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.
Asunto(s)
Anexina A1/fisiología , Virus de la Influenza A/fisiología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Receptores de Formil Péptido/fisiología , Replicación Viral , Animales , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Virus de la Influenza A/patogenicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiologíaRESUMEN
Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3ß-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.
Asunto(s)
Antivirales/farmacología , Cardenólidos/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Antivirales/química , Cardenólidos/química , Humanos , Conformación Molecular , ARN Polimerasa Dependiente del ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular , Células HEK293 , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Modelos Biológicos , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Dominios y Motivos de Interacción de Proteínas , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Eliminación de Secuencia , Transcripción Genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to "open up" and become active already at a low temperature.
Asunto(s)
Núcleo Celular/metabolismo , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Mutación , Proteínas no Estructurales Virales/genética , Células HEK293 , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/fisiología , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas , Temperatura , Proteínas no Estructurales Virales/química , Replicación ViralRESUMEN
Avian influenza A viruses, such as the highly pathogenic avian H5N1 viruses, sporadically enter the human population but often do not transmit between individuals. In rare cases, however, they establish a new lineage in humans. In addition to well-characterized barriers to cell entry, one major hurdle which avian viruses must overcome is their poor polymerase activity in human cells. There is compelling evidence that these viruses overcome this obstacle by acquiring adaptive mutations in the polymerase subunits PB1, PB2, and PA and the nucleoprotein (NP) as well as in the novel polymerase cofactor nuclear export protein (NEP). Recent findings suggest that synthesis of the viral genome may represent the major defect of avian polymerases in human cells. While the precise mechanisms remain to be unveiled, it appears that a broad spectrum of polymerase adaptive mutations can act collectively to overcome this defect. Thus, identification and monitoring of emerging adaptive mutations that further increase polymerase activity in human cells are critical to estimate the pandemic potential of avian viruses.
Asunto(s)
Adaptación Biológica/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/enzimología , Gripe Aviar/virología , Nucleoproteínas/genética , Proteínas Virales/metabolismo , Animales , Aves , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Modelos Biológicos , Mutación/genética , Especificidad de la Especie , Proteínas Virales/genética , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Influenza virus infections represent a major global health problem. The dynamin-like GTPase MX1 is an interferon-dependent antiviral host protein that confers resistance to influenza virus infections. Infection models in mice are an important experimental system to understand the host response and susceptibility to developing severe disease following influenza infections. However, almost all laboratory mouse strains carry a non-functional Mx1 gene whereas humans have a functional MX1 gene. Most studies in mice have been performed with strains carrying a non-functional Mx1 gene. It is therefore very important to investigate the host response in mouse strains with a functional Mx1 gene. RESULTS: Here, we analyzed the host response to influenza virus infections in two congenic mouse strains carrying the functional Mx1 gene from the A2G strain. B6.A2G-Mx1r/r(B6-Mx1r/r) mice are highly resistant to influenza A virus (IAV) H1N1 infections. On the other hand, D2(B6).A2G-Mx1r/r(D2-Mx1r/r) mice, although carrying a functional Mx1 gene, were highly susceptible, exhibited rapid weight loss, and died. We performed gene expression analysis using RNAseq from infected lungs at days 3 and 5 post-infection (p.i.) of both mouse strains to identify genes and pathways that were differentially expressed between the two mouse strains. The susceptible D2-Mx1r/r mice showed a high viral replication already at day 3 p.i. and exhibited a much higher number of differentially expressed genes (DEGs) and many DEGs had elevated expression levels compared to B6-Mx1r/r mice. On the other hand, some DEGs were specifically up-regulated only in B6-Mx1r/r mice at day 3 p.i., many of which were related to host immune response functions. CONCLUSIONS: From these results, we conclude that at early times of infection, D2-Mx1r/r mice showed a very high and rapid replication of the virus, which resulted in lung damage and a hyperinflammatory response leading to death. We hypothesize that the activation of certain immune response genes was missing and that others, especially Mx1, were expressed at a time in D2-Mx1r/r mice when the virus had already massively spread in the lung and were thus not able anymore to protect them from severe disease. Our study represents an important addition to previously published studies in mouse models and contributes to a better understanding of the molecular pathways and genes that protect against severe influenza disease.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Humanos , Ratones , Animales , Subtipo H1N1 del Virus de la Influenza A/genética , Proteínas de Resistencia a Mixovirus/genética , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Infecciones por Orthomyxoviridae/genética , Pulmón , Virus de la Influenza A/genética , Inmunidad Innata , Expresión GénicaRESUMEN
During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1 and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Proteínas Virales/metabolismo , Transcripción Genética , Nucleotidiltransferasas/metabolismo , Replicación Viral , Ubiquitinación , Ubiquitinas/metabolismo , ARN Viral/genéticaRESUMEN
Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Enfermedades de los Porcinos , Animales , Humanos , Porcinos , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/veterinaria , Subtipo H1N1 del Virus de la Influenza A/genética , Virus Reordenados/genética , Virus de la Influenza A/genética , Genómica , Enfermedades de los Porcinos/epidemiología , Gripe Humana/epidemiologíaRESUMEN
SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.
Asunto(s)
Antivirales , COVID-19 , Inflamación , Síndrome Post Agudo de COVID-19 , SARS-CoV-2 , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/virología , Pulmón , Transducción de SeñalRESUMEN
Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.
Asunto(s)
Alphainfluenzavirus , Antivirales , Betainfluenzavirus , Productos Biológicos , Inhibidores Enzimáticos , Metiltransferasas , Caperuzas de ARN , Tubercidina , Replicación Viral , Animales , Humanos , Ratones , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , Replicación Viral/efectos de los fármacos , Alphainfluenzavirus/efectos de los fármacos , Betainfluenzavirus/efectos de los fármacos , Productos Biológicos/química , Productos Biológicos/farmacología , Antivirales/química , Antivirales/farmacología , Tubercidina/análogos & derivados , Tubercidina/farmacología , Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Streptomyces/química , Simulación por Computador , Células A549RESUMEN
The nucleoprotein (NP) of Lassa virus (LASV) strain AV was expressed in a recombinant baculovirus system. The crystal structure of full-length NP was solved at a resolution of 2.45 Å. The overall fold corresponds to that of NP of LASV strain Josiah (Qi, X., Lan, S., Wang, W., Schelde, L. M., Dong, H., Wallat, G. D., Ly, H., Liang, Y., and Dong, C. (2010) Nature 468, 779-783) with a root mean square deviation of 0.67 Å for all atoms (6.3% difference in primary sequence). As the packing in the crystal offers two different trimer architectures for the biological assembly, the quaternary structure of NP in solution was determined by small-angle x-ray scattering and EM. After classification and averaging of >6000 EM raw images, trimeric centrosymmetric structures were obtained, which correspond in size and shape to one trimer in the crystal structure formed around a crystallographic 3-fold rotation axis (symmetric trimer). The symmetric trimer is also a good model for the small-angle x-ray scattering data and could be well embedded into the ab initio model. The N-terminal domain of NP contains a deep nucleotide-binding cavity that has been proposed to bind cellular cap structures for priming viral mRNA synthesis. All residues implicated in m(7)GpppN binding were exchanged, and the transcription/replication phenotype of the NP mutant was tested using a LASV replicon system. None of the mutants showed a specific defect in mRNA expression; most were globally defective in RNA synthesis. In conclusion, we describe the full-length crystal structure and the quaternary structure in solution of LASV NP. The nucleotide-binding pocket of NP could not be assigned a specific role in viral mRNA synthesis.