RESUMEN
Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.
Asunto(s)
Condensados Biomoleculares/virología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Alcaloides de Veratrum/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Línea Celular , Femenino , Humanos , Cuerpos de Inclusión , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Virus Sincitial Respiratorio Humano/fisiología , Factores de Transcripción , Proteínas ViralesRESUMEN
Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria-derived succinate that accumulated both in the respiratory fluids of virus-challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus-triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate-dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Neumonía , Animales , Antivirales/farmacología , Humanos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Ratones , Proteínas de la Nucleocápside , Nucleoproteínas/metabolismo , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Ácido Succínico/uso terapéutico , Replicación ViralRESUMEN
The development of safe and effective vaccines in a record time after the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a remarkable achievement, partly based on the experience gained from multiple viral outbreaks in the past decades. However, the Coronavirus Disease 2019 (COVID-19) crisis also revealed weaknesses in the global pandemic response and large gaps that remain in our knowledge of the biology of coronaviruses (CoVs) and influenza viruses, the 2 major respiratory viruses with pandemic potential. Here, we review current knowns and unknowns of influenza viruses and CoVs, and we highlight common research challenges they pose in 3 areas: the mechanisms of viral emergence and adaptation to humans, the physiological and molecular determinants of disease severity, and the development of control strategies. We outline multidisciplinary approaches and technological innovations that need to be harnessed in order to improve preparedeness to the next pandemic.
Asunto(s)
COVID-19/virología , Gripe Humana/virología , Orthomyxoviridae/fisiología , SARS-CoV-2/fisiología , Animales , Antivirales , COVID-19/terapia , COVID-19/transmisión , Desarrollo de Medicamentos , Evolución Molecular , Humanos , Gripe Humana/terapia , Gripe Humana/transmisión , Orthomyxoviridae/inmunología , SARS-CoV-2/inmunología , Selección Genética , Carga Viral , Vacunas ViralesRESUMEN
Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.
Asunto(s)
Estrógenos no Esteroides , Gripe Humana , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Humanos , Hemaglutininas , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/prevención & control , Orthomyxoviridae/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Antivirales/farmacologíaRESUMEN
PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus-infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2-induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.
Asunto(s)
Infecciones por Orthomyxoviridae/metabolismo , Multimerización de Proteína , Respiración , Transducción de Señal , Proteínas Virales/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Femenino , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Luciferasas/genética , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Infecciones por Orthomyxoviridae/fisiopatología , Infecciones por Orthomyxoviridae/virología , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteínas Virales/genéticaRESUMEN
Along with respiratory tract disease per se, viral respiratory infections can also cause extrapulmonary complications with a potentially critical impact on health. In the present study, we used an experimental model of influenza A virus (IAV) infection to investigate the nature and outcome of the associated gut disorders. In IAV-infected mice, the signs of intestinal injury and inflammation, altered gene expression, and compromised intestinal barrier functions peaked on day 7 postinfection. As a likely result of bacterial component translocation, gene expression of inflammatory markers was upregulated in the liver. These changes occurred concomitantly with an alteration of the composition of the gut microbiota and with a decreased production of the fermentative, gut microbiota-derived products short-chain fatty acids (SCFAs). Gut inflammation and barrier dysfunction during influenza were not attributed to reduced food consumption, which caused in part gut dysbiosis. Treatment of IAV-infected mice with SCFAs was associated with an enhancement of intestinal barrier properties, as assessed by a reduction in the translocation of dextran and a decrease in inflammatory gene expression in the liver. Lastly, SCFA supplementation during influenza tended to reduce the translocation of the enteric pathogen Salmonella enterica serovar Typhimurium and to enhance the survival of doubly infected animals. Collectively, influenza virus infection can remotely impair the gut's barrier properties and trigger secondary enteric infections. The latter phenomenon can be partially countered by SCFA supplementation.
Asunto(s)
Infecciones por Enterobacteriaceae/etiología , Ácidos Grasos Volátiles/biosíntesis , Interacciones Huésped-Patógeno , Virus de la Influenza A/fisiología , Gripe Humana/complicaciones , Gripe Humana/virología , Mucosa Intestinal/metabolismo , Interacciones Microbianas , Susceptibilidad a Enfermedades , Disbiosis , Infecciones por Enterobacteriaceae/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Gripe Humana/metabolismo , Mucosa Intestinal/inmunologíaRESUMEN
The olfactory mucosa, where the first step of odor detection occurs, is a privileged pathway for environmental toxicants and pathogens toward the central nervous system. Indeed, some pathogens can infect olfactory sensory neurons including their axons projecting to the olfactory bulb allowing them to bypass the blood-brain barrier and reach the central nervous system (CNS) through the so-called olfactory pathway. The respiratory syncytial virus (RSV) is a major respiratory tract pathogen but there is growing evidence that RSV may lead to CNS impairments. However, the mechanisms involved in RSV entering into the CNS have been poorly described. In this study, we wanted to explore the capacity of RSV to reach the CNS via the olfactory pathway and to better characterize RSV cellular tropism in the nasal cavity. We first explored the distribution of RSV infectious sites in the nasal cavity by in vivo bioluminescence imaging and a tissue clearing protocol combined with deep-tissue imaging and 3D image analyses. This whole tissue characterization was confirmed with immunohistochemistry and molecular biology approaches. Together, our results provide a novel 3D atlas of mouse nasal cavity anatomy and show that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb. Cover Image for this issue: doi: 10.1111/jnc.14765.
Asunto(s)
Mucosa Olfatoria/inervación , Mucosa Olfatoria/virología , Neuronas Receptoras Olfatorias/virología , Virus Sincitiales Respiratorios , Animales , Sistema Nervioso Central/diagnóstico por imagen , Sistema Nervioso Central/virología , Enfermedades del Sistema Nervioso Central/diagnóstico por imagen , Enfermedades del Sistema Nervioso Central/virología , Femenino , Cabeza/anatomía & histología , Imagenología Tridimensional , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/virología , Bulbo Olfatorio/virología , Mucosa Olfatoria/diagnóstico por imagen , ARN Viral/aislamiento & purificación , Tropismo , Replicación ViralRESUMEN
Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.
Asunto(s)
Antivirales , Péptidos , Conformación Proteica en Hélice alfa , Virus Sincitial Respiratorio Humano , Animales , Antivirales/farmacología , Humanos , Ratones , Péptidos/farmacología , Fosfoproteínas/farmacología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Replicación ViralRESUMEN
Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.
Asunto(s)
Infecciones por Coronavirus/patología , Bulbo Olfatorio/patología , Mucosa Olfatoria/patología , Neumonía Viral/patología , Animales , Betacoronavirus , COVID-19 , Cilios/patología , Infecciones por Coronavirus/fisiopatología , Mesocricetus , Trastornos del Olfato/patología , Trastornos del Olfato/fisiopatología , Bulbo Olfatorio/virología , Mucosa Olfatoria/virología , Neuronas Receptoras Olfatorias/patología , Neuronas Receptoras Olfatorias/virología , Pandemias , Neumonía Viral/fisiopatología , SARS-CoV-2RESUMEN
Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (â¼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.
Asunto(s)
Antivirales , Materiales Biomiméticos , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 2/metabolismo , Nanopartículas , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/metabolismo , Animales , Antivirales/química , Antivirales/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/farmacología , Herpes Simple/metabolismo , Herpes Simple/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/patologíaRESUMEN
Severe bacterial (pneumococcal) infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Disruption of lung tissue integrity during influenza participates in bacterial pulmonary colonization and dissemination out of the lungs. Interleukin-22 (IL-22) has gained considerable interest in anti-inflammatory and anti-infection immunotherapy over the last decade. In the current study, we investigated the effect of exogenous IL-22 delivery on the outcome of pneumococcal superinfection postinfluenza. Our data show that exogenous treatment of influenza virus-infected mice with recombinant IL-22 reduces bacterial dissemination out of the lungs but is without effect on pulmonary bacterial burden. Reduced systemic bacterial dissemination was linked to reinforced pulmonary barrier functions, as revealed by total protein measurement in the bronchoalveolar fluids, intratracheal fluorescein isothiocyanate-dextran tracking, and histological approaches. We describe an IL-22-specific gene signature in the lung tissue of influenza A virus (IAV)-infected (and naive) mice that might explain the observed effects. Indeed, exogenous IL-22 modulates the gene expression profile in a way that suggests reinforcement of tissue integrity. Our results open the way to alternative approaches for limiting postinfluenza bacterial superinfection, particularly, systemic bacterial invasion.
Asunto(s)
Interleucinas/uso terapéutico , Pulmón/inmunología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones Neumocócicas/inmunología , Sobreinfección/inmunología , Animales , Humanos , Inmunoterapia , Hígado/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Transcriptoma , Interleucina-22RESUMEN
PB1-F2 is a virulence factor of influenza A virus (IAV) whose functions remain misunderstood. The different roles of PB1-F2 may be linked to its structural polymorphism and to its propensity to assemble into oligomers and amyloid fibers in the vicinity of the membrane of IAV-infected cells. Here, we monitored the impact of PB1-F2 on the biochemical composition and protein structures of human epithelial pulmonary cells (A549) and monocytic cells (U937) upon IAV infection using synchrotron Fourier-transform infrared (FTIR) and deep UV (DUV) microscopies at the single-cell level. Cells were infected with a wild-type IAV and its PB1-F2 knock-out mutant for analyses at different times post-infection. IR spectra were recorded in each condition and processed to evaluate the change in the component band of the spectra corresponding to the amide I (secondary structure) and the CH stretching region (membrane). The IR spectra analysis revealed that expression of PB1-F2 in U937 cells, but not in A549 cells, results in the presence of a specific ß-aggregate signature. Furthermore, the lipid membrane composition of U937 cells expressing PB1-F2 was also altered in a cell type-dependent manner. Using DUV microscopy and taking advantage of the high content of tryptophan residues in the sequence of PB1-F2 (5/90 aa), we showed that the increase of the autofluorescent signal recorded in monocytic cells could be correlated with the IR detection of ß-aggregates. Altogether, our results constitute an important step forward in the understanding of the cell type-dependent function of PB1-F2.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Agregado de Proteínas , Proteínas Virales/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/virología , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/genética , Microscopía Fluorescente , Células U937 , Proteínas Virales/genéticaRESUMEN
Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.
Asunto(s)
Antivirales/farmacología , Péptidos/farmacología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Proteínas Virales de Fusión/química , Internalización del Virus/efectos de los fármacos , Administración Intranasal , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antivirales/síntesis química , Sitios de Unión , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Péptidos/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/química , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Replicación Viral/efectos de los fármacosRESUMEN
PB1-F2 is a viral protein encoded by influenza A viruses (IAVs). PB1-F2 is implicated in virulence by triggering immune cell apoptosis and enhancing inflammation. To obtain an insight into the molecular mechanisms of PB1-F2-mediated virulence, we used the yeast two-hybrid approach to find new PB1-F2 cellular interactors. This allowed us to identify calcium-binding and coiled-coil domain 2 (CALCOCO2, also known as NDP52) as a binding partner of PB1-F2. Binding of PB1-F2 to CALCOCO2 was confirmed by pull-down. Surface plasmon resonance binding experiments enabled us to estimate the dissociation constant (Kd) of the two partners to be around 20 nM. Using bioinformatics tools, we designed a CALCOCO2 interaction map based on previous knowledge and showed a strong connection between this protein and the type I interferon production pathways and the I-κB kinase/NF-κB signalling pathway. NF-κB reporter assays in which CALCOCO2, MAVS and PB1-F2 were co-expressed showed a cooperation of these three proteins to increase the inflammatory response. By contrast, PB1-F2 inhibits the TBK1-dependent activation of an ISRE reporter plasmid. We also demonstrated that the signal transducer TRAF6 is implicated in the enhancement of NF-κB activity mediated by PB1-F2/CALCOCO2 binding. Altogether, this report provides evidence of an interaction link between PB1-F2 and human proteins, and allows a better understanding of the involvement of PB1-F2 in the pathologic process mediated by IAV.
Asunto(s)
Interacciones Huésped-Patógeno , Inmunidad Innata , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Biología Computacional , Humanos , Cinética , Unión Proteica , Mapeo de Interacción de Proteínas , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos HíbridosRESUMEN
UNLABELLED: The influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four viable mutants with single-codon deletions were generated; all of them exhibited a ts phenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serial in vitro passages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts mutants that could be used to design novel attenuated vaccines. IMPORTANCE: In order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA-PB1 dimer to the nucleus, where viral replication occurs. These ts deletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.
Asunto(s)
Codón , Virus de la Influenza A/fisiología , Proteínas Mutantes/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Eliminación de Secuencia , Proteínas Virales/metabolismo , Replicación Viral , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Inestabilidad Genómica , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Ratones Endogámicos BALB C , Viabilidad Microbiana , Proteínas Mutantes/genética , Infecciones por Orthomyxoviridae/virología , ARN Polimerasa Dependiente del ARN/genética , Temperatura , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Proteínas Virales/genéticaRESUMEN
Highly pathogenic influenza A viruses (IAV) infections represent a serious threat to humans due to their considerable morbidity and mortality capacities. A good understanding of the molecular mechanisms responsible for the acute lung injury observed during this kind of infection is essential to design adapted therapies. In the current study, using an unbiased transcriptomic approach, we compared the host-responses of mice infected with two different subtypes of IAV: H1N1 vs. H5N1. The host-response comparison demonstrated a clear difference between the transcriptomic profiles of H1N1- and H5N1-infected mice despite identical survival kinetics and similar viral replications. The ontological analysis of the two transcriptomes showed two probable causes of death: induction of an immunopathological state of the lung for the H1N1 strain vs. development of respiratory dysfunction in the case of the H5N1 IAV. Finally, a clear signature responsible for lung edema was specifically associated with the H5N1 infection. We propose a potential mechanism of edema development based on predictive bioinformatics tools.
Asunto(s)
Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Animales , Análisis por Conglomerados , Epistasis Genética , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Infecciones por Orthomyxoviridae/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Carga ViralRESUMEN
UNLABELLED: The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and ß-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines. IMPORTANCE: By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H1N1 del Virus de la Influenza A/efectos de la radiación , Mutación , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de la radiación , Transporte Activo de Núcleo Celular , Animales , Dicroismo Circular , Femenino , Prueba de Complementación Genética , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica/efectos de la radiación , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN Polimerasa Dependiente del ARN/química , Temperatura , Proteínas Virales/químicaRESUMEN
PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 µM and from 5 nM to 0.5 µM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent.
Asunto(s)
Técnicas Electroquímicas , Virus de la Influenza A/fisiología , Proteínas Virales/análisis , Anticuerpos Monoclonales/inmunología , Benzotiazoles , Técnicas Biosensibles , Línea Celular Tumoral , Humanos , Microscopía de Fuerza Atómica , Multimerización de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Tiazoles/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.
Asunto(s)
Dibenzocicloheptenos , Piridinas , Infecciones por Virus Sincitial Respiratorio , Animales , Femenino , Ratones , Reposicionamiento de Medicamentos , Piperidinas/farmacología , Piperidinas/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/químicaRESUMEN
Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence.