RESUMEN
Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory senescence-associated secretory phenotype (SASP), is a cause of aging. In senescent cells, cytoplasmic chromatin fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. Promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of nuclear factor κB (NF-κB) target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in the regulation of SASP.
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Alphaviruses are a large group of re-emerging arthropod-borne RNA viruses. The compact viral RNA genomes harbor diverse structures that facilitate replication. These structures can be recognized by antiviral cellular RNA-binding proteins, including DExD-box (DDX) helicases, that bind viral RNAs to control infection. The full spectrum of antiviral DDXs and the structures that are recognized remain unclear. Genetic screening identified DDX39A as antiviral against the alphavirus chikungunya virus (CHIKV) and other medically relevant alphaviruses. Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm inhibiting alphavirus replication, independent of the canonical interferon pathway. Biochemically, DDX39A binds to CHIKV genomic RNA, interacting with the 5' conserved sequence element (5'CSE), which is essential for the antiviral activity of DDX39A. Altogether, DDX39A relocalization and binding to a conserved structural element in the alphavirus genomic RNA attenuates infection, revealing a previously unknown layer to the cellular control of infection.
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Fiebre Chikungunya , Virus Chikungunya , Humanos , Virus Chikungunya/genética , Línea Celular , Fiebre Chikungunya/metabolismo , ARN Helicasas/metabolismo , Replicación Viral/genética , ARN Viral/genética , ARN Viral/metabolismo , Antivirales/farmacología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismoRESUMEN
Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.
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Virus Chikungunya , Interferón Tipo I , ARN Largo no Codificante , Antivirales/farmacología , Virus Chikungunya/genética , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , ARN Largo no Codificante/genética , ARN Viral/genética , Replicación Viral/genéticaRESUMEN
The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.
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Antivirales , Evaluación Preclínica de Medicamentos , Nucleósidos , Pirimidinas , SARS-CoV-2 , Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , COVID-19/virología , Línea Celular , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Nucleósidos/análogos & derivados , Nucleósidos/farmacología , Pirimidinas/farmacología , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.
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Proliferación Celular , Senescencia Celular , Ensamble y Desensamble de Cromatina , Cromatina/enzimología , Fibroblastos/enzimología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Proliferación Celular/genética , Senescencia Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Represión Epigenética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/genética , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
Rift Valley fever virus (RVFV) is an encephalitic bunyavirus that can infect neurons in the brain. There are no approved therapeutics that can protect from RVFV encephalitis. Innate immunity, the first line of defense against infection, canonically antagonizes viruses through interferon signaling. We found that interferons did not efficiently protect primary cortical neurons from RVFV, unlike other cell types. To identify alternative neuronal antiviral pathways, we screened innate immune ligands and discovered that the TLR2 ligand Pam3CSK4 inhibited RVFV infection, and other bunyaviruses. Mechanistically, we found that Pam3CSK4 blocks viral fusion, independent of TLR2. In a mouse model of RVFV encephalitis, Pam3CSK4 treatment protected animals from infection and mortality. Overall, Pam3CSK4 is a bunyavirus fusion inhibitor active in primary neurons and the brain, representing a new approach toward the development of treatments for encephalitic bunyavirus infections.
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Lipopéptidos , Neuronas , Fiebre del Valle del Rift , Virus de la Fiebre del Valle del Rift , Animales , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Ratones , Lipopéptidos/farmacología , Fiebre del Valle del Rift/virología , Fiebre del Valle del Rift/prevención & control , Neuronas/metabolismo , Neuronas/virología , Ratones Endogámicos C57BL , Humanos , Inmunidad Innata/efectos de los fármacos , Encefalitis Viral/virología , Encefalitis Viral/inmunología , Encefalitis Viral/prevención & control , Encefalitis Viral/tratamiento farmacológico , Antivirales/farmacologíaRESUMEN
The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in the deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.
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Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.
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Empalme Alternativo , Leucemia Mieloide Aguda/genética , Mutación , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Genotipo , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.
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COVID-19/prevención & control , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Compuestos de Litio/uso terapéutico , Adulto , Anciano , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Humanos , Compuestos de Litio/farmacología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Estudios RetrospectivosRESUMEN
MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB-SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB-SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein-protein interactions or promote degradation of meiosis-specific proteins.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Meiosis/fisiología , Testículo/metabolismo , Animales , Fertilidad/fisiología , Células HEK293 , Humanos , Masculino , RatonesRESUMEN
Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.
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Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Replicación del ADN , Desoxirribonucleasas/metabolismo , Células HeLa , Chaperonas de Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismoRESUMEN
INTRODUCTION: The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown. METHODS: We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types. RESULTS: Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis. CONCLUSIONS: Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.
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Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/genética , Naftoquinonas/farmacología , Empalme Alternativo , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Camptotecina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Daño del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Etopósido/farmacología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Células MCF-7 , Paclitaxel/farmacología , Receptor ErbB-2/metabolismo , Survivin , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target. SIGNIFICANCE: The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.
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Ácidos y Sales Biliares , Carcinoma de Células Renales , Colesterol , Homeostasis , Neoplasias Renales , Humanos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/genética , Ácidos y Sales Biliares/metabolismo , Colesterol/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/genética , Animales , Ratones , Triterpenos Pentacíclicos , Línea Celular Tumoral , Apoptosis , Proliferación Celular , Triterpenos/farmacología , Carcinogénesis/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory Senescence-Associated Secretory Phenotype (SASP), is a cause of aging. In senescent cells, Cytoplasmic Chromatin Fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. PML protein organizes PML nuclear bodies (NBs), also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of NF-κB target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in regulation of SASP.
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Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.
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Daño del ADN , Exodesoxirribonucleasas , Fosfoproteínas , Animales , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratones , Reparación del ADN por Recombinación , Fenotipo , Mutación , Drosophila/genética , Envejecimiento/genética , Envejecimiento/metabolismo , Femenino , Drosophila melanogaster/genética , Masculino , Enfermedades de la Retina , Enfermedades Vasculares , Enfermedades Desmielinizantes del Sistema Nervioso Central HereditariasRESUMEN
The cellular DNA-damage response is a signaling network that is vigorously activated by cytotoxic DNA lesions, such as double-strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which modulates this process by phosphorylating key players in these pathways. A long-standing question in this field is whether DSB formation affects chromatin condensation. Here, we show that DSB formation is followed by ATM-dependent chromatin relaxation. ATM's effector in this pathway is the protein KRAB-associated protein (KAP-1, also known as TIF1beta, KRIP-1 or TRIM28), previously known as a corepressor of gene transcription. In response to DSB induction, KAP-1 is phosphorylated in an ATM-dependent manner on Ser 824. KAP-1 is phosphorylated exclusively at the damage sites, from which phosphorylated KAP-1 spreads rapidly throughout the chromatin. Ablation of the phosphorylation site of KAP-1 leads to loss of DSB-induced chromatin decondensation and renders the cells hypersensitive to DSB-inducing agents. Knocking down KAP-1, or mimicking a constitutive phosphorylation of this protein, leads to constitutive chromatin relaxation. These results suggest that chromatin relaxation is a fundamental pathway in the DNA-damage response and identify its primary mediators.
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Proteínas de Ciclo Celular/fisiología , Cromatina/metabolismo , Daño del ADN , Proteínas de Unión al ADN/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/fisiología , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Microscopía Fluorescente , Mutación/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos Tripartito , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Cinostatina/farmacologíaRESUMEN
The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.
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Rubella is a highly contagious viral infection that usually causes a mild disease in children and adults. However, infection during pregnancy can result in a fetal or newborn death or congenital rubella syndrome (CRS), a constellation of permanent birth defects including cataracts, heart defects, and sensorineural deafness. The live-attenuated rubella vaccine has been highly effective, with the Americas declared free of endemic rubella transmission in 2015. However, rubella remains a significant problem worldwide and the leading cause of vaccine-preventable birth defects globally. Thus, elimination of rubella and CRS is a goal of the World Health Organization. No specific therapeutics are approved for the rubella virus. Therefore, we set out to identify whether existing small molecules may be repurposed for use against rubella virus infection. Thus, we performed a high-throughput screen for small molecules active against rubella virus in human respiratory cells and identified two nucleoside analogs, NM107 and AT-527, with potent antiviral activity. Furthermore, we found that combining these nucleoside analogs with inhibitors of host nucleoside biosynthesis had synergistic antiviral activity. These studies open the door to new potential approaches to treat rubella infections.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-ß treatment. However, upon treatment with IFN-ß, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-ß-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.
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COVID-19 , Interferón Tipo I , Humanos , SARS-CoV-2/metabolismo , Proteínas Virales/metabolismo , Interferones , Inmunidad InnataRESUMEN
SARS-CoV-2 emerged, and is evolving to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling activated only in bystander cells. This balance of innate activation and viral evasion has important consequences, but the pathways involved are incompletely understood. Here we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons, and thus permissivity to infection. Mechanistically, autophagy genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, upon loss of autophagy increased MAVS overcomes ORF9b-mediated antagonism suppressing infection. This has led to the evolution of SARS-CoV-2 variants to express higher levels of ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of autophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.