RESUMEN
Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia , Autofagia , Humanos , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Animales , Autofagosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Membrana Celular/metabolismoRESUMEN
Reprogramming lipid metabolic pathways is a critical feature of activating immune responses to infection. However, how these reconfigurations occur is poorly understood. Our previous screen to identify cellular deubiquitylases (DUBs) activated during influenza virus infection revealed Usp25 as a prominent hit. Here, we show that Usp25-deleted human lung epithelial A549 cells display a >10-fold increase in pathogenic influenza virus production, which was rescued upon reconstitution with the wild type but not the catalytically deficient (C178S) variant. Proteomic analysis of Usp25 interactors revealed a strong association with Erlin1/2, which we confirmed as its substrate. Newly synthesized Erlin1/2 were degraded in Usp25-/- or Usp25C178S cells, activating Srebp2, with increased cholesterol flux and attenuated TLR3-dependent responses. Our study therefore defines the function of a deubiquitylase that serves to restrict a range of viruses by reprogramming lipid biosynthetic flux to install appropriate inflammatory responses.
Asunto(s)
Colesterol , Ubiquitina Tiolesterasa , Virosis , Humanos , Lípidos , Pulmón/metabolismo , Proteómica , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Virosis/genética , Virosis/metabolismo , Colesterol/metabolismoRESUMEN
Conjugation of the Atg8 (autophagy related 8) family of ubiquitin-like proteins to phospholipids of the phagophore is a hallmark of macroautophagy/autophagy. Consequently, Atg8 family members, especially LC3B, are commonly used as a marker of autophagosomes. However, the Atg8 family of proteins are not found solely attached to double-membrane autophagosomes. In non-canonical Atg8-family protein lipidation they become conjugated to single membranes. We have shown that this process is triggered by recruitment of ATG16L1 by the vacuolar-type H+-translocating ATPase (V-ATPase) proton pump, suggesting a role for pH sensing in recruitment of Atg8-family proteins to single membranes.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Autofagia , Proteínas Asociadas a Microtúbulos , ATPasas de Translocación de Protón , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , ATPasas de Translocación de Protón/metabolismoRESUMEN
Determining how hematopoietic stem and progenitor cells (HSPCs) can be infected by viruses is necessary to understand and predict how the immune system will drive the host response. We present here a protocol to analyze the capacity of SARS-CoV-2 to infect different subsets of human HSPCs, inlcuding procedures for SARS-CoV-2 production and titration, isolation of human HSPCs from different sources (bone marrow, umbilical cord, or peripheral blood), and quantification of SARS-Cov-2 infection capacity by RT-qPCR and colony forming unit assay. For complete details on the use and execution of this protocol, please refer to Huerga Encabo et al. (2021).
Asunto(s)
Médula Ósea/virología , Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/virología , Ensayo de Unidades Formadoras de Colonias/métodos , Sangre Fetal/virología , Células Madre Hematopoyéticas/virología , SARS-CoV-2/aislamiento & purificación , COVID-19/patología , Células Madre Hematopoyéticas/patología , HumanosRESUMEN
Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.
Asunto(s)
Proteínas Relacionadas con la Autofagia , Lipoilación , Proteínas Asociadas a Microtúbulos , Autofagosomas/genética , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Sistemas CRISPR-Cas , Células HCT116 , Células HEK293 , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Salmonella/genética , Salmonella/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismoRESUMEN
In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Interferencia de ARN , Virus ARN/fisiología , ARN Viral/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Células Madre/enzimología , Células Madre/virología , Empalme Alternativo , Animales , Encéfalo/enzimología , Encéfalo/virología , Línea Celular , ARN Helicasas DEAD-box/química , Humanos , Inmunidad Innata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Organoides/enzimología , Organoides/virología , Infecciones por Virus ARN/enzimología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Virus ARN/genética , Virus ARN/inmunología , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/química , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , Replicación Viral , Virus Zika/genética , Virus Zika/inmunología , Virus Zika/fisiología , Infección por el Virus Zika/enzimología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , ARN Helicasas/antagonistas & inhibidores , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Chlorocebus aethiops , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , ARN Helicasas/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Suramina/farmacología , Células Vero , Proteínas no Estructurales Virales/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.
Asunto(s)
Antivirales/química , Antivirales/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Benzoatos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Chlorocebus aethiops , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Holoenzimas/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Suramina/farmacología , Células Vero , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismoRESUMEN
SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Endorribonucleasas/antagonistas & inhibidores , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Animales , Chlorocebus aethiops , Endorribonucleasas/aislamiento & purificación , Endorribonucleasas/metabolismo , Pruebas de Enzimas , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Técnicas In Vitro , Cinética , Naftoquinonas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/química , Soluciones , Células Vero , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismoRESUMEN
The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Compuestos de Anilina/farmacología , Animales , Benzamidas/farmacología , Chlorocebus aethiops , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/aislamiento & purificación , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Sinergismo Farmacológico , Pruebas de Enzimas , Flavinas/farmacología , Transferencia Resonante de Energía de Fluorescencia , Furanos/farmacología , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Naftalenos/farmacología , Fenantrenos/farmacología , Quinonas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/antagonistas & inhibidores , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Animales , Ácido Aurintricarboxílico/farmacología , Chlorocebus aethiops , Pruebas de Enzimas , Exorribonucleasas/metabolismo , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Patulina/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Azoles/farmacología , Chlorocebus aethiops , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/aislamiento & purificación , Proteasas 3C de Coronavirus/metabolismo , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Isoindoles , Leupeptinas/farmacología , Compuestos de Organoselenio/farmacología , Peptidomiméticos , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Proteínas no Estructurales Virales/metabolismoRESUMEN
The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/química , Clorobencenos/farmacología , Chlorocebus aethiops , Pruebas de Enzimas , Exorribonucleasas/genética , Exorribonucleasas/aislamiento & purificación , Exorribonucleasas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Indazoles/farmacología , Indenos/farmacología , Indoles/farmacología , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Nitrilos/farmacología , Fenotiazinas/farmacología , Purinas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Especificidad por Sustrato , Trifluperidol/farmacología , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.
Asunto(s)
COVID-19 , Genoma Viral , Motivos de Nucleótidos , Pliegue del ARN , ARN Viral , SARS-CoV-2/fisiología , Replicación Viral , Animales , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , ARN Viral/genética , ARN Viral/metabolismo , Células VeroRESUMEN
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/genética , Autofagosomas/patología , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Virus de la Influenza A/patogenicidad , Macrólidos/farmacología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Monensina/farmacología , Fagocitosis , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.
Asunto(s)
COVID-19/inmunología , Hemo/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Bilirrubina/metabolismo , Biliverdina/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos , Humanos , Sueros Inmunes , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidadRESUMEN
We document here that intensive care COVID-19 patients suffer a profound decline in hemoglobin levels but show an increase of circulating nucleated red cells, suggesting that SARS-CoV-2 infection either directly or indirectly induces stress erythropoiesis. We show that ACE2 expression peaks during erythropoiesis and renders erythroid progenitors vulnerable to infection by SARS-CoV-2. Early erythroid progenitors, defined as CD34-CD117+CD71+CD235a-, show the highest levels of ACE2 and constitute the primary target cell to be infected during erythropoiesis. SARS-CoV-2 causes the expansion of colony formation by erythroid progenitors and can be detected in these cells after 2 weeks of the initial infection. Our findings constitute the first report of SARS-CoV-2 infectivity in erythroid progenitor cells and can contribute to understanding both the clinical symptoms of severe COVID-19 patients and how the virus can spread through the circulation to produce local inflammation in tissues, including the bone marrow.
Asunto(s)
COVID-19/virología , Células Precursoras Eritroides/virología , Eritropoyesis/fisiología , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , Línea Celular , Chlorocebus aethiops , Células Precursoras Eritroides/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/virología , Células VeroRESUMEN
The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.
RESUMEN
Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/inmunología , Inmunidad Humoral , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , COVID-19/sangre , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/química , Zoonosis Virales/sangre , Zoonosis Virales/inmunología , Adulto JovenRESUMEN
Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.