RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de SinaisRESUMO
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.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Humanos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Animais , Autofagossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismoRESUMO
A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Transporte/fisiologia , Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Repetições WD40 , Animais , Apresentação de Antígeno , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células Dendríticas/metabolismo , Endossomos/metabolismo , Feminino , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
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.
Assuntos
COVID-19 , Genoma Viral , Motivos de Nucleotídeos , Dobramento de RNA , RNA Viral , SARS-CoV-2/fisiologia , Replicação Viral , Animais , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Células VeroRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Holoenzimas/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , RNA Helicases/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/metabolismoRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Endorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Chlorocebus aethiops , Endorribonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Ensaios Enzimáticos , Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Cinética , Naftoquinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Soluções , Células Vero , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismoRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Chlorocebus aethiops , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/isolamento & purificação , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Sinergismo Farmacológico , Ensaios Enzimáticos , Flavinas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Furanos/farmacologia , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Naftalenos/farmacologia , Fenantrenos/farmacologia , Quinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Animais , Ácido Aurintricarboxílico/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/metabolismo , Fluorescência , Ensaios de Triagem em Larga Escala , Patulina/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
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.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Azóis/farmacologia , Chlorocebus aethiops , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Isoindóis , Leupeptinas/farmacologia , Compostos Organosselênicos/farmacologia , Peptidomiméticos , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismoRESUMO
The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.
Assuntos
Antivirais/química , Cardiovirus/enzimologia , Enterovirus Humano B/enzimologia , Poliovirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Sítios de Ligação , Chlorocebus aethiops , Células HeLa , Humanos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.
Assuntos
Antivirais/farmacologia , Enterovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Carbazóis/farmacologia , Proteínas de Transporte/genética , Clopentixol/farmacologia , Dibucaína/farmacologia , Enterovirus/genética , Fluoxetina/farmacologia , Fumarato de Formoterol/farmacologia , Células HeLa , Humanos , Rhinovirus/efeitos dos fármacos , Rhinovirus/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
UNLABELLED: Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIß (phosphatidylinositol-4-kinase IIIß) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIß to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIß through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIß. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIß to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIß recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIß via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIß. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIß, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIß to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE: A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these "replication organelles" depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are formed and which cellular components are key players in this process. GBF1/Arf1 and ACBD3 have been proposed to contribute to the recruitment of the essential lipid-modifying enzyme PI4KIIIß to enterovirus replication organelles. Here we show that the enterovirus CVB3 recruits PI4KIIIß by a mechanism independent of both GBF1/Arf1 and ACBD3. This study shows that the strategy employed by coxsackievirus to recruit PI4KIIIß to replication organelles is far more complex than initially anticipated.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Enterovirus Humano B/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Replicação Viral , Animais , Linhagem Celular , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Humanos , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)--a selective serotonin reuptake inhibitor--inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound.
Assuntos
Proteínas de Transporte/metabolismo , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/metabolismo , Enterovirus Humano D/efeitos dos fármacos , Enterovirus Humano D/metabolismo , Fluoxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Proteínas não Estruturais Virais/metabolismo , Proteínas de Transporte/genética , Enterovirus Humano B/genética , Enterovirus Humano D/genética , Proteínas não Estruturais Virais/genéticaRESUMO
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.
Assuntos
Colesterol , Ubiquitina Tiolesterase , Viroses , Humanos , Lipídeos , Pulmão/metabolismo , Proteômica , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Viroses/genética , Viroses/metabolismo , Colesterol/metabolismoRESUMO
The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (M(pro)) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV M(pro) is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-ß-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV M(pro) autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln↓(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of M(pro), resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative M(pro) autoprocessing sites has not been described previously for other nidovirus M(pro) domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales.
Assuntos
Coronaviridae/enzimologia , Serina Proteases/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Domínio Catalítico , Análise Mutacional de DNA , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina Proteases/genética , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Proteínas Associadas aos Microtúbulos , ATPases Translocadoras de Prótons , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , ATPases Translocadoras de Prótons/metabolismoRESUMO
Nidoviruses employ unique strategies to replicate and express their exceptionally large RNA genomes. The viruses use a variety of enzymes to synthesize, modify and process an extensive set of viral RNAs of both genome and subgenome length, including RNA polymerase, primase, helicase, ribose 2'-O and guanosine-N7 methyltransferases and several types of nuclease activities. In this review, the recent progress in the structural and functional characterization of nidovirus nuclease activities is discussed, focusing on a nidovirus-wide conserved uridylate-specific endoribonuclease, NendoU, and a 3'-to-5' exoribonuclease called ExoN. The latter enzyme is related to members of the DEDD exoribonuclease superfamily and conserved in all nidovirus families with genome sizes approaching 30 kilobases. Recent evidence implicates ExoN in reduced mutation rates during viral RNA replication and, possibly, superior fidelity of nidovirus replicases, leading to the suggestion that ExoN may be a key factor in the expansion of nidovirus genomes to sizes not seen in other RNA viruses.
Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Nidovirales/química , Nidovirales/fisiologia , Replicação Viral , Animais , Genoma Viral , Humanos , Nidovirales/genética , RNA Viral/genéticaRESUMO
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).