RESUMEN
Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.
Asunto(s)
COVID-19/virología , Furina/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Línea Celular , Endosomas/metabolismo , Furina/genética , Expresión Génica , Humanos , Evasión Inmune , Interferones/metabolismo , Lisosomas/enzimología , Coactivadores de Receptor Nuclear/genética , Isoformas de Proteínas , Proteolisis , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Pseudotipado Viral , Internalización del VirusRESUMEN
Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , COVID-19/genética , COVID-19/fisiopatología , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/fisiología , Regiones no Traducidas 5' , Células A549 , Animales , COVID-19/enzimología , COVID-19/inmunología , Quirópteros/genética , Quirópteros/virología , Coronaviridae/enzimología , Coronaviridae/genética , Coronaviridae/fisiología , Endorribonucleasas/metabolismo , Humanos , Interferones/inmunología , Isoenzimas/genética , Isoenzimas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo de Nucleótido Simple , Prenilación de Proteína , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Viral/química , ARN Viral/genética , Retroelementos , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Replicación ViralRESUMEN
The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19/diagnóstico , COVID-19/virología , Genética Inversa , SARS-CoV-2/genética , Células A549 , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Chlorocebus aethiops , Codón , Humanos , Hidrazonas/farmacología , Ratones , Morfolinas/farmacología , Sistemas de Lectura Abierta , Plásmidos/genética , Pirimidinas/farmacología , Serina Endopeptidasas/metabolismo , Células Vero , Proteínas Virales/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.