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Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.
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COVID-19 , Quirópteros , Inmunidad Innata , SARS-CoV-2 , Replicación Viral , Animales , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Quirópteros/virología , Quirópteros/inmunología , COVID-19/transmisión , COVID-19/virología , COVID-19/inmunología , Ratones , Cricetinae , Evasión Inmune , Células Epiteliales/virología , Células Epiteliales/inmunología , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Coronavirus/inmunología , Coronavirus/genética , Coronavirus/clasificación , Coronavirus/fisiología , Coronavirus/patogenicidad , Línea Celular , FemeninoRESUMEN
Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude, neutralization, and duration of anti-receptor binding domain antibodies compared to Alum vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.
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Vacunas contra la COVID-19 , COVID-19 , Inmunidad Humoral , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Ratones , COVID-19/prevención & control , COVID-19/inmunología , Porosidad , Femenino , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Materiales Biocompatibles/química , Ratones Endogámicos BALB C , Linfocitos B/inmunología , SARS-CoV-2/inmunología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & controlRESUMEN
Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.
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Administración Intranasal , Antivirales , Neomicina , SARS-CoV-2 , Animales , Neomicina/farmacología , Neomicina/administración & dosificación , Ratones , Humanos , Antivirales/farmacología , Antivirales/administración & dosificación , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/prevención & control , Mucosa Nasal/inmunología , Mucosa Nasal/virología , Mucosa Nasal/efectos de los fármacos , Modelos Animales de Enfermedad , Tratamiento Farmacológico de COVID-19 , Mesocricetus , Femenino , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunologíaRESUMEN
The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection.
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Regulación de la Expresión Génica , Inmunidad Innata , Proteínas de la Membrana , FN-kappa B , Proteínas Oncogénicas , Proteínas de Unión al ARN , Animales , Humanos , Sistemas CRISPR-Cas , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Células HEK293 , Inmunidad Innata/genética , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Transducción de Señal , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunologíaRESUMEN
The persistent murine norovirus strain MNVCR6 is a model for human norovirus and enteric viral persistence. MNVCR6 causes chronic infection by directly infecting intestinal tuft cells, rare chemosensory epithelial cells. Although MNVCR6 induces functional MNV-specific CD8+ T cells, these lymphocytes fail to clear infection. To examine how tuft cells promote immune escape, we interrogated tuft cell interactions with CD8+ T cells by adoptively transferring JEDI (just EGFP death inducing) CD8+ T cells into Gfi1b-GFP tuft cell reporter mice. Unexpectedly, some intestinal tuft cells partially resisted JEDI CD8+ T cell-mediated killing-unlike Lgr5+ intestinal stem cells and extraintestinal tuft cells-despite seemingly normal antigen presentation. When targeting intestinal tuft cells, JEDI CD8+ T cells predominantly adopted a T resident memory phenotype with decreased effector and cytotoxic capacity, enabling tuft cell survival. JEDI CD8+ T cells neither cleared nor prevented MNVCR6 infection in the colon, the site of viral persistence, despite targeting a virus-independent antigen. Ultimately, we show that intestinal tuft cells are relatively resistant to CD8+ T cells independent of norovirus infection, representing an immune-privileged niche that can be leveraged by enteric microbes.
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Linfocitos T CD8-positivos , Norovirus , Ratones , Humanos , Animales , Células en Penacho , Norovirus/fisiología , Privilegio Inmunológico , IntestinosRESUMEN
Chronic inflammation and dysregulated repair mechanisms after epithelial damage have been implicated in chronic obstructive pulmonary disease (COPD). However, the lack of ex vivo-models that accurately reflect multicellular lung tissue hinders our understanding of epithelial-mesenchymal interactions in COPD. Through a combination of transcriptomic and proteomic approaches applied to a sophisticated in vitro iPSC-alveolosphere with fibroblasts model, epithelial-mesenchymal crosstalk was explored in COPD and following SARS-CoV-2 infection. These experiments profiled dynamic changes at single-cell level of the SARS-CoV-2-infected alveolar niche that unveiled the complexity of aberrant inflammatory responses, mitochondrial dysfunction, and cell death in COPD, which provides deeper insights into the accentuated tissue damage/inflammation/remodeling observed in patients with SARS-CoV-2 infection. Importantly, this 3D system allowed for the evaluation of ACE2-neutralizing antibodies and confirmed the potency of this therapy to prevent SARS-CoV-2 infection in the alveolar niche. Thus, iPSC-alveolosphere cultured with fibroblasts provides a promising model to investigate disease-specific mechanisms and to develop novel therapeutics.
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COVID-19 , Células Madre Pluripotentes Inducidas , Enfermedad Pulmonar Obstructiva Crónica , Humanos , SARS-CoV-2 , Proteómica , Inmunoterapia , InflamaciónRESUMEN
Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2 , Cromatina , Proteómica , Senescencia Celular , Células Gigantes , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape we conducted a gain of function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including JADE3 a protein involved in directing the histone acetyltransferase HBO1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Interestingly, expression of the closely related paralogues JADE1 and JADE2 are unable to restrict influenza A virus infection, suggesting a distinct function of JADE3. We identify both shared and unique transcriptional signatures between uninfected cells expressing JADE3 and JADE2. These data provide a framework for understanding the overlapping and distinct functions of the JADE family of paralogues. Specifically, we find that JADE3 expression activates the NF-kB signaling pathway, consistent with an antiviral function. Therefore, we propose JADE3, but not JADE1 or JADE2, activates an antiviral genetic program involving the NF-kB pathway to restrict influenza A virus infection.
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Diverse mammalian species display susceptibility to and infection with SARS-CoV-2. Potential SARS-CoV-2 spillback into rodents is understudied despite their host role for numerous zoonoses and human proximity. We assessed exposure and infection among white-footed mice (Peromyscus leucopus) in Connecticut, USA. We observed 1% (6/540) wild-type neutralizing antibody seroprevalence among 2020-2022 residential mice with no cross-neutralization of variants. We detected no SARS-CoV-2 infections via RT-qPCR, but identified non-SARS-CoV-2 betacoronavirus infections via pan-coronavirus PCR among 1% (5/468) of residential mice. Sequencing revealed two divergent betacoronaviruses, preliminarily named Peromyscus coronavirus-1 and -2. Both belong to the Betacoronavirus 1 species and are ~90% identical to the closest known relative, Porcine hemagglutinating encephalomyelitis virus. Low SARS-CoV-2 seroprevalence suggests white-footed mice may not be sufficiently susceptible or exposed to SARS-CoV-2 to present a long-term human health risk. However, the discovery of divergent, non-SARS-CoV-2 betacoronaviruses expands the diversity of known rodent coronaviruses and further investigation is required to understand their transmission extent.
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Norovirus is a leading cause of epidemic viral gastroenteritis, with no currently approved vaccines or antivirals. Murine norovirus (MNoV) is a well-characterized model of norovirus pathogenesis in vivo, and persistent strains exhibit lifelong intestinal infection. Interferon-λ (IFN-λ) is a potent antiviral that rapidly cures MNoV. We previously demonstrated that IFN-λ signaling in intestinal epithelial cells (IECs) controls persistent MNoV, and here demonstrate that IFN-λ acts on tuft cells, the exclusive site of MNoV persistence, to limit infection. While interrogating the source of IFN-λ to regulate MNoV, we confirmed that MDA5-MAVS signaling, required for IFN-λ induction to MNoV in vitro, controls persistent MNoV in vivo. We demonstrate that MAVS in IECs and not immune cells controls MNoV. MAVS in nonsusceptible enterocytes, but not in tuft cells, restricts MNoV, implicating noninfected cells as the IFN-λ source. Our findings indicate that host sensing of MNoV is distinct from cellular tropism, suggesting intercellular communication between IECs for antiviral signaling induction in uninfected bystander cells.
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Infecciones por Enterovirus , Norovirus , Animales , Ratones , Enterocitos , Células Epiteliales , Transducción de Señal , Antivirales/farmacología , Interferón lambdaRESUMEN
COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
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Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.
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Neoplasias Pulmonares , Animales , Humanos , Ensamble y Desensamble de Cromatina , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Cromatina , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores ErbB/genética , Mutación , Mamíferos/genética , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents.
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Caliciviridae , Norovirus , Ratones , Humanos , Animales , Norovirus/genética , Antivirales/farmacología , Caliciviridae/genética , Genoma , Mamíferos/genéticaRESUMEN
Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.
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COVID-19 , Proteínas de Transferencia de Fosfolípidos , SARS-CoV-2 , Animales , Humanos , Ratones , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Quirópteros , COVID-19/inmunología , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Secuenciación del Exoma , Hepatocitos/inmunología , Hepatocitos/metabolismo , Interferón gamma/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Fusión de Membrana , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/inmunología , Proteínas de Transferencia de Fosfolípidos/metabolismo , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Internalización del VirusRESUMEN
Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Histona Demetilasas/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/metabolismoRESUMEN
Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.
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ARN , Terbio , Conformación de Ácido Nucleico , ARN/metabolismo , Terbio/metabolismo , Terbio/farmacología , Motivos de Nucleótidos , CationesRESUMEN
Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.
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COVID-19 , Internalización del Virus , Animales , Humanos , Enzima Convertidora de Angiotensina 2 , COVID-19/genética , COVID-19/metabolismo , Dipeptidil Peptidasa 4 , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , SARS-CoV-2/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Quinasas DyrKRESUMEN
Passive immunization with nirsevimab protects infants from severe RSV disease without impairing the immune response to natural infection.
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Infecciones por Virus Sincitial Respiratorio , Lactante , Humanos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Inmunización PasivaRESUMEN
Norovirus (NoV) is the leading global cause of viral gastroenteritis. Young children bear the highest burden of disease and play a key role in viral transmission throughout the population. However, which host factors contribute to age-associated variability in NoV severity and shedding are not well-defined. The murine NoV (MNoV) strain CR6 causes persistent infection in adult mice and targets intestinal tuft cells. Here we find that natural transmission of CR6 from infected dams occurred only in juvenile mice. Direct oral CR6 inoculation of wild-type neonatal mice led to accumulation of viral RNA in the ileum and prolonged shedding in the stool that was replication-independent. This viral exposure induced both innate and adaptive immune responses including interferon-stimulated gene expression and MNoV-specific antibody responses. Interestingly, viral uptake depended on passive ileal absorption of luminal virus, a process blocked by cortisone acetate administration, which prevented ileal viral RNA accumulation. Neonates lacking interferon signalling in haematopoietic cells were susceptible to productive infection, viral dissemination and lethality, which depended on the canonical MNoV receptor CD300LF. Together, our findings reveal developmentally associated aspects of persistent MNoV infection, including distinct tissue and cellular tropism, mechanisms of interferon regulation and severity of infection in the absence of interferon signalling. These emphasize the importance of defining viral pathogenesis phenotypes across the developmental spectrum and highlight passive viral uptake as an important contributor to enteric infections in early life.
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Infecciones por Caliciviridae , Norovirus , Ratones , Animales , Interferones , Intestinos , Intestino Delgado/metabolismoRESUMEN
Identification of host determinants of coronavirus infection informs mechanisms of viral pathogenesis and can provide new drug targets. Here we demonstrate that mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complexes, specifically canonical BRG1/BRM-associated factor (cBAF) complexes, promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and represent host-directed therapeutic targets. The catalytic activity of SMARCA4 is required for mSWI/SNF-driven chromatin accessibility at the ACE2 locus, ACE2 expression and virus susceptibility. The transcription factors HNF1A/B interact with and recruit mSWI/SNF complexes to ACE2 enhancers, which contain high HNF1A motif density. Notably, small-molecule mSWI/SNF ATPase inhibitors or degraders abrogate angiotensin-converting enzyme 2 (ACE2) expression and confer resistance to SARS-CoV-2 variants and a remdesivir-resistant virus in three cell lines and three primary human cell types, including airway epithelial cells, by up to 5 logs. These data highlight the role of mSWI/SNF complex activities in conferring SARS-CoV-2 susceptibility and identify a potential class of broad-acting antivirals to combat emerging coronaviruses and drug-resistant variants.