RESUMEN
Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin ß RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.
Asunto(s)
Antivirales , Virus de la Influenza A , ARN Polimerasa Dependiente del ARN , Anticuerpos de Dominio Único , Replicación Viral , Anticuerpos de Dominio Único/inmunología , Humanos , Antivirales/farmacología , Virus de la Influenza A/inmunología , Animales , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Gripe Humana/inmunología , Gripe Humana/virología , Células HEK293 , Perros , Células de Riñón Canino Madin DarbyRESUMEN
The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Proteínas Recombinantes de Fusión , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Antivirales/farmacología , Humanos , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.
Asunto(s)
Enfermedades de los Caballos , Subtipo H3N8 del Virus de la Influenza A , Infecciones por Orthomyxoviridae , Vacunas , Animales , Aminoácidos/genética , Genómica , Caballos , Subtipo H3N8 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/veterinaria , Reproducibilidad de los Resultados , Análisis de Secuencia/veterinaria , Factores de VirulenciaRESUMEN
We introduce multiple interface string alignment (MISA), a visualization tool to display coherently various sequence and structure based statistics at protein-protein interfaces (SSE elements, buried surface area, ΔASA , B factor values, etc). The amino acids supporting these annotations are obtained from Voronoi interface models. The benefit of MISA is to collate annotated sequences of (homologous) chains found in different biological contexts, that is, bound with different partners or unbound. The aggregated views MISA/SSE, MISA/BSA, MISA/ΔASA, and so forth, make it trivial to identify commonalities and differences between chains, to infer key interface residues, and to understand where conformational changes occur upon binding. As such, they should prove of key relevance for knowledge-based annotations of protein databases such as the Protein Data Bank. Illustrations are provided on the receptor binding domain of coronaviruses, in complex with their cognate partner or (neutralizing) antibodies. MISA computed with a minimal number of structures complement and enrich findings previously reported. The corresponding package is available from the Structural Bioinformatics Library (http://sbl.inria.frand https://sbl.inria.fr/doc/Multiple_interface_string_alignment-user-manual.html).
Asunto(s)
Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/química , Secuencia de Aminoácidos , Biología Computacional , Bases de Datos de Proteínas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Análisis de Secuencia de Proteína , Interfaz Usuario-ComputadorRESUMEN
Picobirnaviridae is a family of viruses with bi-segmented (rarely unsegmented) dsRNA genomes comprising about 4.4 kbp in total, with small, non-enveloped spherical virions. The family includes one genus (Picobirnavirus) grouping three genetic clusters with high sequence variability, two defined by viruses infecting vertebrates and a third with viruses found in invertebrates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Picobirnaviridae, which is available at www.ictv.global/report/picobirnaviridae.
Asunto(s)
Picobirnavirus/clasificación , Picobirnavirus/genética , Enfermedades de los Animales/virología , Animales , Invertebrados/virología , Picobirnavirus/aislamiento & purificación , Infecciones por Virus ARN/veterinaria , Vertebrados/virologíaRESUMEN
Birnaviridae is a family of viruses with bi-segmented dsRNA genomes totalling about 6 kbp forming icosahedral, non-enveloped virions. The family includes four genera, members of three of which (Aquabirnavirus, Avibirnavirus and Blosnavirus) infect vertebrates (excluding mammals), whereas members of the fourth genus (Entomobirnavirus) infect insects. Each genus includes 1-3 species. Infectious pancreatic necrosis virus of salmonids and infectious bursal disease virus of poultry are two economically important birnaviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Birnaviridae, which is available at www.ictv.global/report/birnaviridae.
Asunto(s)
Birnaviridae/clasificación , ARN Viral/genética , Virión/ultraestructura , Animales , Birnaviridae/genética , Birnaviridae/aislamiento & purificación , Birnaviridae/ultraestructura , Insectos/virología , Vertebrados/virologíaRESUMEN
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
PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.
Asunto(s)
Amiloide/toxicidad , Liposomas/metabolismo , Proteínas Virales/toxicidad , Antiinfecciosos/farmacología , Bacillus subtilis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Escherichia coli/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/patogenicidad , Virus de la Influenza A/ultraestructura , Liposomas/ultraestructura , Pruebas de Sensibilidad Microbiana , Permeabilidad , Agregado de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Virales/químicaRESUMEN
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
Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.
Asunto(s)
Virus de la Influenza A/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Sitios de Unión , Dicroismo Circular , Cristalización , Virus de la Influenza A/química , Virus de la Influenza A/ultraestructura , Mutación , Tamaño de la Partícula , Fosforilación , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Viral/química , Ribonucleoproteínas/química , Proteínas Virales/químicaRESUMEN
BACKGROUND: Birnaviruses form a distinct family of double-stranded RNA viruses infecting animals as different as vertebrates, mollusks, insects and rotifers. With such a wide host range, they constitute a good model for studying the adaptation to the host. Additionally, several lines of evidence link birnaviruses to positive strand RNA viruses and suggest that phylogenetic analyses may provide clues about transition. RESULTS: We characterized the genome of a birnavirus from the rotifer Branchionus plicalitis. We used X-ray structures of RNA-dependent RNA polymerases and capsid proteins to obtain multiple structure alignments that allowed us to obtain reliable multiple sequence alignments and we employed "advanced" phylogenetic methods to study the evolutionary relationships between some positive strand and double-stranded RNA viruses. We showed that the rotifer birnavirus genome exhibited an organization remarkably similar to other birnaviruses. As this host was phylogenetically very distant from the other known species targeted by birnaviruses, we revisited the evolutionary pathways within the Birnaviridae family using phylogenetic reconstruction methods. We also applied a number of phylogenetic approaches based on structurally conserved domains/regions of the capsid and RNA-dependent RNA polymerase proteins to study the evolutionary relationships between birnaviruses, other double-stranded RNA viruses and positive strand RNA viruses. CONCLUSIONS: We show that there is a good correlation between the phylogeny of the birnaviruses and that of their hosts at the phylum level using the RNA-dependent RNA polymerase (genomic segment B) on the one hand and a concatenation of the capsid protein, protease and ribonucleoprotein (genomic segment A) on the other hand. This correlation tends to vanish within phyla. The use of advanced phylogenetic methods and robust structure-based multiple sequence alignments allowed us to obtain a more accurate picture (in terms of probability of the tree topologies) of the evolutionary affinities between double-stranded RNA and positive strand RNA viruses. In particular, we were able to show that there exists a good statistical support for the claims that dsRNA viruses are not monophyletic and that viruses with permuted RdRps belong to a common evolution lineage as previously proposed by other groups. We also propose a tree topology with a good statistical support describing the evolutionary relationships between the Picornaviridae, Caliciviridae, Flaviviridae families and a group including the Alphatetraviridae, Nodaviridae, Permutotretraviridae, Birnaviridae, and Cystoviridae families.
Asunto(s)
Evolución Molecular , Virus ARN/genética , Rotíferos/virología , Secuencia de Aminoácidos , Animales , Genoma Viral , Especificidad del Huésped , Filogenia , Virus ARN/clasificación , Virus ARN/fisiología , Virus ARN/efectos de la radiación , ARN Bicatenario/genética , Rotíferos/clasificación , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antivirales/farmacología , Naproxeno/farmacología , Nucleoproteínas/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antivirales/química , Sitios de Unión , Perros , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/química , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Naproxeno/química , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Mutación Puntual , Unión Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 A X-ray structure of a picobirnavirus--an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties.
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Picobirnavirus/química , Picobirnavirus/ultraestructura , Proteínas Virales/química , Virión/química , Virión/ultraestructura , Secuencia de Aminoácidos , Animales , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Picobirnavirus/fisiología , Procesamiento Proteico-Postraduccional , Virión/fisiología , Internalización del VirusRESUMEN
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.
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Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/inmunología , Transcriptoma , Proteínas Virales/metabolismo , Animales , Apoptosis , Muerte Celular , Quimiotaxis , Femenino , Eliminación de Gen , Regulación Viral de la Expresión Génica , Ingeniería Genética , Interferón beta/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Neutrófilos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/genética , Transcripción Genética , Proteínas Virales/genética , VirulenciaRESUMEN
After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.
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Infecciones por Bunyaviridae/veterinaria , Enfermedades Transmisibles Emergentes/veterinaria , Orthobunyavirus/fisiología , Rumiantes , Animales , Infecciones por Bunyaviridae/diagnóstico , Infecciones por Bunyaviridae/epidemiología , Infecciones por Bunyaviridae/etiología , Enfermedades Transmisibles Emergentes/diagnóstico , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/etiología , Europa (Continente)/epidemiología , Orthobunyavirus/clasificación , Orthobunyavirus/genética , Orthobunyavirus/patogenicidadRESUMEN
The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-ß gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-ß expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-ß expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-ß expression and found that PB1-F2-mediated IFN-ß induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.
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Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Interferón beta/biosíntesis , Mucosa Respiratoria/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Apoptosis/inmunología , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Gripe Humana/metabolismo , Interferón beta/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas Virales/genética , Virulencia/genéticaRESUMEN
Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.