RESUMO
Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.
Assuntos
Anticorpos Antivirais , Imunidade nas Mucosas , Vírus da Influenza A , Vacinas contra Influenza , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Animais , Camundongos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Feminino , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Vacinas Combinadas/imunologia , Vacinas Combinadas/administração & dosagem , Humanos , Adenoviridae/imunologia , Adenoviridae/genética , Vetores GenéticosRESUMO
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.
Assuntos
Dibenzocicloeptenos , Piridinas , Infecções por Vírus Respiratório Sincicial , Animais , Feminino , Camundongos , Reposicionamento de Medicamentos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/químicaRESUMO
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.
Assuntos
COVID-19/diagnóstico , Hibridização in Situ Fluorescente/métodos , RNA Viral/genética , SARS-CoV-2/genética , Replicação Viral/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/farmacologia , COVID-19/virologia , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Hibridização In Situ/métodos , Microscopia Eletrônica/métodos , RNA Viral/ultraestrutura , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Células Vero , Liberação de Vírus/efeitos dos fármacos , Liberação de Vírus/genética , Liberação de Vírus/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Tratamento Farmacológico da COVID-19RESUMO
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.
Assuntos
Condensados Biomoleculares/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linhagem Celular , Feminino , Humanos , Corpos de Inclusão , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus Sincicial Respiratório Humano/fisiologia , Fatores de Transcrição , Proteínas ViraisRESUMO
Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.
Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/virologia , Pulmão/imunologia , Células-Tronco Mesenquimais/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Animais , Humanos , Pulmão/citologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , OvinosRESUMO
Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.
Assuntos
Anticorpos Antivirais/administração & dosagem , Imunização Passiva , Imunoglobulina G/administração & dosagem , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Modelos Animais de Doenças , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Pulmão/efeitos dos fármacos , Pulmão/virologia , Testes de Neutralização , Infecções por Vírus Respiratório Sincicial/virologia , Conchas Nasais/efeitos dos fármacos , Conchas Nasais/virologia , Proteínas Virais de Fusão/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus-host interactions and related acute respiratory disease, at the level of the entire lung. METHODS: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. RESULTS: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. CONCLUSIONS: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.
Assuntos
Imageamento Tridimensional , Pulmão/patologia , Pulmão/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Animais , Modelos Animais de Doenças , Células Epiteliais/virologia , Corpos de Inclusão Viral/patologia , Camundongos , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Replicação ViralRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a current pandemic worldwide. This virus can reach all organs and disturbs the immune system, leading to a cytokine storm in severe forms. We aimed to report cutaneous features among coronavirus disease 2019 (COVID-19) hospitalized patients. METHODS: We performed a cross-sectional study on 1 given day among all patients hospitalized in acute care for COVID-19 and included all patients with cutaneous features. Follow-up 48 hours later was obtained. RESULTS: Among 59 adult patients hospitalized on the day of the study in an infectious diseases ward for SARS-CoV-2 infection who were confirmed by molecular assay and/or radiological findings (computed tomography scan), 40 were included. Several cutaneous manifestations were found: macular exanthema (80%), face edema (32%), livedo (13%), urticarial rash (8%), purpura (5%), oral lichenoid lesions (33%), and conjunctivitis (18%). Cutaneous biopsy was performed in 17 patients. Histological findings showed mast cell hyperplasia (100%), superficial perivascular infiltrate of lymphocytes (94%), and superficial edema (47%) consistent with capillary leak. CONCLUSIONS: Various dermatological signs can be encountered during COVID-19. A macular rash was the most frequent. All cutaneous features could be related to a vascular leak process.
RESUMO
Respiratory syncytial virus (RSV) is the prevalent pathogen of lower respiratory tract infections in children. The presence of neonatal regulatory B lymphocytes (nBreg) has been associated with a poor control of RSV infection in human newborns and with bronchiolitis severity. So far, little is known about how nBreg may contribute to neonatal immunopathology to RSV. We tracked nBreg in neonatal BALB/c mice and we investigated their impact on lung innate immunity, especially their crosstalk with alveolar macrophages (AMs) upon RSV infection. We showed that the colonization by nBreg during the first week of life is a hallmark of neonatal lung whereas this population is almost absent in adult lung. This particular period of age when nBreg are abundant corresponds to the same period when RSV replication in lungs fails to generate a type-I interferons (IFN-I) response and is not contained. When neonatal AMs are exposed to RSV in vitro, they produce IFN-I that in turn enhances IL-10 production by nBreg. IL-10 reciprocally can decrease IFN-I secretion by AMs. Thus, our work identified nBreg as an important component of neonatal lungs and pointed out new immunoregulatory interactions with AMs in the context of RSV infection.
Assuntos
Linfócitos B Reguladores/imunologia , Interleucina-10/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Conchas Nasais/imunologia , Animais , Animais Recém-Nascidos , Subpopulações de Linfócitos B/imunologia , Células Cultivadas , Imunidade Inata , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Interleucina-10/metabolismo , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Baço/imunologia , Replicação ViralRESUMO
The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.
Assuntos
Antivirais/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanabenzo/análogos & derivados , Animais , Antivirais/uso terapêutico , Linhagem Celular , Vírus de DNA/efeitos dos fármacos , Vírus de DNA/fisiologia , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Infecções por Poxviridae/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/fisiologia , Coelhos , Infecções Tumorais por Vírus/tratamento farmacológico , Replicação Viral/efeitos dos fármacosRESUMO
Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Epitopos/química , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão/química , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/química , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Clonagem Molecular , Desenho Assistido por Computador , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imunização/métodos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Palivizumab/química , Palivizumab/imunologia , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/biossíntese , Vacinas contra Vírus Sincicial Respiratório/genética , Homologia Estrutural de Proteína , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
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.
Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Proteínas Virais de Fusão/química , Internalização do Vírus/efeitos dos fármacos , Administração Intranasal , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antivirais/síntese química , Sítios de Ligação , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Replicação Viral/efeitos dos fármacosRESUMO
Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infections in children, yet no vaccine is available. The sole licensed preventive treatment against RSV is composed of a monoclonal neutralizing antibody (palivizumab), which targets a conformational epitope located on the fusion protein (F). Palivizumab reduces the burden of bronchiolitis but does not prevent infection. Thus, the development of RSV vaccines remains a priority. We previously evaluated nanorings formed by RSV nucleoprotein (N) as an RSV vaccine, as well as an immunostimulatory carrier for heterologous antigens. Here, we linked the palivizumab-targeted epitope (called FsII) to N, to generate N-FsII-nanorings. Intranasal N-FsII immunization elicited anti-F antibodies in mice that were non-neutralizing in vitro. Nevertheless, RSV-challenged animals were better protected against virus replication than mice immunized with N-nanorings, especially in the upper airways. In conclusion, an N-FsII-focused vaccine is an attractive candidate combining N-specific cellular immunity and F-specific antibodies for protection.
Assuntos
Epitopos , Nanopartículas , Vacinas contra Vírus Sincicial Respiratório , Vírus Sinciciais Respiratórios , Proteínas Virais de Fusão , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Camundongos , Palivizumab , Infecções por Vírus Respiratório Sincicial/prevenção & controle , SigmodontinaeRESUMO
UNLABELLED: The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy. IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N(0)) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Mapeamento de Interação de Proteínas , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Centrifugação/métodos , Cricetinae , Análise Mutacional de DNA , Mutagênese Sítio-Dirigida , Ligação Proteica , Vírus Sincicial Respiratório Humano/genética , Ressonância de Plasmônio de Superfície , Proteínas Estruturais Virais/genéticaRESUMO
Respiratory syncytial virus (RSV) is the most important cause of severe lower-respiratory tract disease in calves and young children, yet no human vaccine nor efficient curative treatments are available. Here we describe a recombinant human RSV reverse genetics system in which the red fluorescent protein (mCherry) or the firefly luciferase (Luc) genes are inserted into the RSV genome. Expression of mCherry and Luc are correlated with infection rate, allowing the monitoring of RSV multiplication in cell culture. Replication of the Luc-encoding virus in living mice can be visualized by bioluminescent imaging, bioluminescence being detected in the snout and lungs of infected mice after nasal inoculation. We propose that these recombinant viruses are convenient and valuable tools for screening of compounds active against RSV, and can be used as an extremely sensitive readout for studying effects of antiviral therapeutics in living mice.
Assuntos
Microscopia de Fluorescência , Vírus Sinciciais Respiratórios/fisiologia , Animais , Anticorpos Antivirais/imunologia , Antivirais/química , Sequência de Bases , Replicação do DNA , DNA Complementar/metabolismo , Feminino , Fluorescência , Vetores Genéticos , Humanos , Luciferases/metabolismo , Luminescência , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Vírus Sincicial Respiratório Humano , Vírus Sinciciais Respiratórios/patogenicidade , Proteínas Virais de Fusão/genética , Replicação Viral , Proteína Vermelha FluorescenteRESUMO
The underlying mechanisms of the epidemiological association between influenza virus infections and Neisseria meningitidis invasive infections are not fully understood. Here we report that adhesion of N. meningitidis to human Hec-1-B epithelial cells is enhanced by influenza A virus (IAV) infection. A potential role of the viral neuraminidase (NA) in facilitating meningococcal adhesion to influenza virus-infected epithelial cells was examined. Expression of a recombinant IAV NA in Hec-1-B human epithelial cells increased the adhesion of strains of N. meningitidis belonging to the sialic acid-containing capsular serogroups B, C, and W135 but not to the mannosamine phosphate-containing capsular serogroup A. Adhesion enhancement was not observed with an inactive NA mutant or in the presence of an NA inhibitor (zanamivir). Furthermore, purified IAV NA was shown to cleave sialic acid-containing capsular polysaccharides of N. meningitidis. On the whole, our findings suggest that a direct interaction between the NA of IAV and the capsule of N. meningitidis enhances bacterial adhesion to cultured epithelial cells, most likely through cleavage of capsular sialic acid-containing polysaccharides. A better understanding of the association between IAV and invasive meningococcal infections should help to set up improved control strategies against these seasonal dual viral-bacterial infections.
Assuntos
Aderência Bacteriana , Cápsulas Bacterianas/fisiologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Neisseria meningitidis/fisiologia , Neuraminidase/fisiologia , Ácidos Siálicos/fisiologia , Linhagem Celular Tumoral , Células Epiteliais/microbiologia , Humanos , Neuraminidase/genéticaRESUMO
Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Influenza Humana/enzimologia , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Western Blotting , Linhagem Celular , Cães , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/virologia , Proteínas do Nucleocapsídeo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio de Placa ViralAssuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/enzimologia , Neuraminidase/antagonistas & inibidores , Oseltamivir/farmacologia , Sequência de Bases , Citocinas , DNA Viral/análise , Farmacorresistência Viral/fisiologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Estações do Ano , Proteínas Supressoras de Tumor , Replicação Viral/fisiologiaRESUMO
Influenza A viruses are responsible for significant morbidity and mortality after bone marrow transplantation. Here we report failure of inhaled zanamivir treatment in a bone-marrow transplant recipient with pneumonia caused by an influenza A (H1N1) virus, although the influenza viruses isolated from bronchoalveolar lavages before and after treatment were clearly found to be sensitive to zanamivir using cell-based and enzymatic assays. Subsequent oral treatment with oseltamivir allowed complete recovery. Poor bioavailability of zanamivir in the peripheral lungs might have been limiting treatment efficacy in such an immunocompromised patient.