RESUMEN
The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
Asunto(s)
Antígenos Virales/genética , Betacoronavirus/patogenicidad , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Células A549 , Animales , Antígenos Virales/inmunología , Betacoronavirus/genética , Betacoronavirus/inmunología , Sitios de Unión , Bovinos , Chlorocebus aethiops , Cricetinae , Perros , Glicosilación , Células HEK293 , Células HeLa , Humanos , Macaca mulatta , Células de Riñón Canino Madin Darby , Ratones , Células RAW 264.7 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Porcinos , Células Vero , Virulencia/genéticaRESUMEN
Here, we describe the discovery of a naturally occurring human antibody (Ab), FluA-20, that recognizes a new site of vulnerability on the hemagglutinin (HA) head domain and reacts with most influenza A viruses. Structural characterization of FluA-20 with H1 and H3 head domains revealed a novel epitope in the HA trimer interface, suggesting previously unrecognized dynamic features of the trimeric HA protein. The critical HA residues recognized by FluA-20 remain conserved across most subtypes of influenza A viruses, which explains the Ab's extraordinary breadth. The Ab rapidly disrupted the integrity of HA protein trimers, inhibited cell-to-cell spread of virus in culture, and protected mice against challenge with viruses of H1N1, H3N2, H5N1, or H7N9 subtypes when used as prophylaxis or therapy. The FluA-20 Ab has uncovered an exceedingly conserved protective determinant in the influenza HA head domain trimer interface that is an unexpected new target for anti-influenza therapeutics and vaccines.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae , Animales , Perros , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.
Asunto(s)
Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunoglobulina G/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae , Adulto , Animales , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Masculino , Ratones , Persona de Mediana Edad , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.
Asunto(s)
Uniones Estrechas/genética , Proteínas de la Zonula Occludens/genética , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-2/genética , Animales , Sitios de Unión/genética , Adhesión Celular/genética , Polaridad Celular/genética , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Dominios PDZ/genética , Fosfoproteínas/genética , Fosforilación/genética , Unión Proteica/genética , Transducción de Señal/genética , Uniones Estrechas/metabolismo , Proteínas de la Zonula Occludens/química , Proteínas de la Zonula Occludens/ultraestructura , Proteína de la Zonula Occludens-1/química , Proteína de la Zonula Occludens-1/ultraestructura , Proteína de la Zonula Occludens-2/química , Proteína de la Zonula Occludens-2/ultraestructura , Dominios Homologos src/genéticaRESUMEN
Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor-mediated innate immunity1,2. Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid-inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3' untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all-trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells.
Asunto(s)
COVID-19/inmunología , Proteína 58 DEAD Box/inmunología , Pulmón/inmunología , Receptores Inmunológicos/inmunología , SARS-CoV-2/inmunología , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Perros , Células HEK293 , Humanos , Interferón Tipo I/inmunología , Interferones/inmunología , Pulmón/virología , Células de Riñón Canino Madin Darby , Enfermedad Pulmonar Obstructiva Crónica/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Células Sf9 , Transducción de Señal/inmunología , Células Vero , Proteínas Virales/inmunología , Interferón lambdaRESUMEN
Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis , Competencia Celular , Células Epiteliales/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pulmonares/metabolismo , Lesiones Precancerosas/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Antígenos CD/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Perros , Células Epiteliales/inmunología , Células Epiteliales/patología , Células HaCaT , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Células de Riñón Canino Madin Darby , Mecanotransducción Celular , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Lesiones Precancerosas/genética , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Células RAW 264.7 , Receptores Inmunológicos/genética , Estrés Mecánico , Quinasas Asociadas a rho/metabolismoRESUMEN
The fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds, mediating long-range signaling. Here, we show that propagation of membrane tension occurs quickly in cell-attached blebs but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. Perturbations to tension propagate diffusively, with a diffusion coefficient Dσ â¼0.024 µm2/s in HeLa cells. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus, membrane tension is not a mediator of long-range intracellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.
Asunto(s)
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Canales Iónicos/metabolismo , Modelos Biológicos , Transducción de Señal/fisiología , Animales , Perros , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Ratones , Células 3T3 NIH , RatasRESUMEN
B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunidad Innata , Gripe Humana/inmunología , Interleucina-4/genética , Células Asesinas Naturales/inmunología , Infección por el Virus Zika/inmunología , Animales , Pollos , Perros , Centro Germinal/citología , Humanos , Interleucina-4/metabolismo , Macaca , Macrófagos/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BLRESUMEN
Type III interferon (IFN-λ) is important for innate immune protection at mucosal surfaces and has therapeutic benefit against influenza A virus (IAV) infection. However, the mechanisms by which IFN-λ programs adaptive immune protection against IAV are undefined. Here we found that IFN-λ signaling in dendritic cell (DC) populations was critical for the development of protective IAV-specific CD8+ T cell responses. Mice lacking the IFN-λ receptor (Ifnlr1-/-) had blunted CD8+ T cell responses relative to wild type and exhibited reduced survival after heterosubtypic IAV re-challenge. Analysis of DCs revealed IFN-λ signaling directed the migration and function of CD103+ DCs for development of optimal antiviral CD8+ T cell responses, and bioinformatic analyses identified IFN-λ regulation of a DC IL-10 immunoregulatory network. Thus, IFN-λ serves a critical role in bridging innate and adaptive immunity from lung mucosa to lymph nodes to program DCs to direct effective T cell immunity against IAV.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Virus de la Influenza A/inmunología , Interferón gamma/inmunología , Infecciones por Orthomyxoviridae/inmunología , Receptores de Interferón/inmunología , Animales , Línea Celular , Perros , Femenino , Inmunidad Innata/inmunología , Interleucina-10/inmunología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interferón/genética , Receptor de Interferón gammaRESUMEN
Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Membrana Sinovial/inmunología , Inmunidad Adaptativa/inmunología , Animales , Artritis Reumatoide/inmunología , Línea Celular , Perros , Humanos , Mediadores de Inflamación/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratas Wistar , Transducción de Señal/inmunologíaRESUMEN
Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.
Asunto(s)
Uniones Estrechas , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-1/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/química , Animales , Membrana Celular/metabolismo , Membrana Celular/química , Humanos , Unión Proteica , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Humectabilidad , Termodinámica , Perros , Mutación , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/químicaRESUMEN
Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
Asunto(s)
Anticuerpos Antivirales/inmunología , Dominio Catalítico/inmunología , Virus de la Influenza B/inmunología , Neuraminidasa/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Perros , Femenino , Células HEK293 , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Leucocitos Mononucleares/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Infecciones por Orthomyxoviridae/inmunologíaRESUMEN
KRas is a major proto-oncogene product whose signaling activity depends on its level of enrichment on the plasma membrane (PM). This PM localization relies on posttranslational prenylation for membrane affinity, while PM specificity has been attributed to electrostatic interactions between negatively charged phospholipids in the PM and basic amino-acids in the C terminus of KRas. By measuring kinetic parameters of KRas dynamics in living cells with a cellular-automata-based data-fitting approach in realistic cell-geometries, we show that charge-based specificity is not sufficient to generate PM enrichment in light of the total surface area of endomembranes. Instead, mislocalized KRas is continuously sequestered from endomembranes by cytosolic PDEδ to be unloaded in an Arl2-dependent manner to perinuclear membranes. Electrostatic interactions then trap KRas at the recycling endosome (RE), from where vesicular transport restores enrichment on the PM. This energy driven reaction-diffusion cycle explains how small molecule targeting of PDEδ affects the spatial organization of KRas.
Asunto(s)
Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas ras/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Animales , Perros , Proteínas de Unión al GTP/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Células de Riñón Canino Madin Darby , Proto-Oncogenes MasRESUMEN
The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were 'preferentially' expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons.
Asunto(s)
Citocinas/inmunología , Expresión Génica/inmunología , Interleucinas/inmunología , Infecciones por Rotavirus/inmunología , Animales , Células CACO-2 , Línea Celular , Chlorocebus aethiops , Citocinas/genética , Citocinas/farmacología , Perros , Sinergismo Farmacológico , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Immunoblotting , Interleucinas/genética , Interleucinas/farmacología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/virología , Células de Riñón Canino Madin Darby , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Receptores de Citocinas/genética , Receptores de Citocinas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Rotavirus/genética , Infecciones por Rotavirus/virología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Células Vero , Interleucina-22RESUMEN
The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Gripe Humana/inmunología , Orthomyxoviridae/fisiología , ARN Helicasas/metabolismo , ARN Polimerasa II/metabolismo , Degeneraciones Espinocerebelosas/genética , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Citocinas/metabolismo , ADN Helicasas , Perros , Regulación hacia Abajo , Humanos , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Análisis por Micromatrices , Enzimas Multifuncionales , ARN Helicasas/genética , ARN Polimerasa II/genética , ARN Interferente Pequeño/genética , Ataxias Espinocerebelosas/congénito , Células Vero , Replicación Viral/genéticaRESUMEN
Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1-3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR-CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Replicación del ADN , Muerte Celular Regulada , Fase S , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Puntos de Control del Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Perros , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Células de Riñón Canino Madin Darby , Interferencia de ARNRESUMEN
The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Marcación de Gen/métodos , Estabilidad del ARN , Virus ARN/enzimología , ARN Viral/metabolismo , Células A549 , Animales , Proteínas Asociadas a CRISPR/genética , Chlorocebus aethiops , Perros , Escherichia coli/enzimología , Escherichia coli/genética , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Virus ARN/genética , ARN Viral/genética , Células VeroRESUMEN
Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.
Asunto(s)
Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Antivirales/farmacología , Antivirales/química , Química Farmacéutica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Cristalografía por Rayos X/métodos , Química Clic/métodos , Animales , Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby , Inhibidores de Proteínas Virales de Fusión/farmacología , Inhibidores de Proteínas Virales de Fusión/química , PerrosRESUMEN
An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Análisis de la Célula Individual/métodos , Animales , Células de Riñón Canino Madin Darby , Virus de la Influenza A/genética , Perros , Replicación ViralRESUMEN
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.