RESUMEN
The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.
Asunto(s)
Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Recuento de Colonia Microbiana , Microbioma Gastrointestinal/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Inmunidad , Ratones Endogámicos C57BL , Sulfuros/metabolismo , Taurina/farmacologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.
Asunto(s)
Infecciones por Coronavirus/genética , Estudio de Asociación del Genoma Completo , SARS-CoV-2/fisiología , Células A549 , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Coronavirus Humano 229E/fisiología , Infecciones por Coronavirus/virología , Coronavirus Humano NL63/fisiología , Coronavirus Humano OC43/fisiología , Técnicas de Inactivación de Genes , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Mapeo de Interacción de ProteínasRESUMEN
Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
Asunto(s)
Infecciones por Coronavirus/genética , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/inmunología , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Coronavirus/clasificación , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/inmunología , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Células Vero , Internalización del VirusRESUMEN
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
Asunto(s)
COVID-19/genética , Infecciones por Coronavirus/genética , Coronavirus/fisiología , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Animales , Vías Biosintéticas/efectos de los fármacos , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Colesterol/biosíntesis , Colesterol/metabolismo , Análisis por Conglomerados , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resfriado Común/genética , Resfriado Común/virología , Coronavirus/clasificación , Infecciones por Coronavirus/virología , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Ratones , Fosfatidilinositoles/biosíntesis , Células Vero , Internalización del Virus/efectos de los fármacos , Replicación ViralRESUMEN
To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
Asunto(s)
COVID-19/genética , COVID-19/virología , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Vías Biosintéticas , COVID-19/metabolismo , Colesterol/biosíntesis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endosomas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes/métodos , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Interferencia de ARN , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Carga Viral/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Antibiotic tolerance, the capacity of genetically susceptible bacteria to survive the lethal effects of antibiotic treatment, plays a critical and underappreciated role in the disease burden of bacterial infections. Here, we take a pathogen-by-pathogen approach to illustrate the clinical significance of antibiotic tolerance and discuss how the physiology of specific pathogens in their infection environments impacts the mechanistic underpinnings of tolerance. We describe how these insights are leading to the development of species-specific therapeutic strategies for targeting antibiotic tolerance and highlight experimental platforms that are enabling us to better understand the complexities of drug-tolerant pathogens in in vivo settings.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Tolerancia a Medicamentos , Animales , Bacterias/clasificación , Infecciones Bacterianas/microbiología , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Especificidad de la EspecieRESUMEN
Interleukin 2 (IL-2) promotes Foxp3+ regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some Treg cells downregulated CD25, upregulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.
Asunto(s)
Interleucina-2/farmacología , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Interleucina-2/administración & dosificación , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Orthomyxoviridae/fisiología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Asunto(s)
Coronavirus Humano OC43/fisiología , Procesamiento Postranscripcional del ARN/genética , SARS-CoV-2/fisiología , Replicación Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Línea Celular , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas de la Nucleocápside , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.
Asunto(s)
Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , ARN Bacteriano/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Recuento de Colonia Microbiana , Endocitosis/efectos de los fármacos , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Helicobacter pylori/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Níquel/farmacología , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.
Asunto(s)
Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasa/genética , Receptores de Factores de Crecimiento/genética , Proteínas Virales/genética , Corticoesteroides/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Células CACO-2 , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/metabolismo , SARS-CoV-2 , Transducción de Señal , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.
Asunto(s)
Adaptación Fisiológica/inmunología , Anticuerpos/inmunología , Linfocitos B/inmunología , Tracto Gastrointestinal/inmunología , Inmunoglobulina A Secretora/inmunología , Microbiota/inmunología , Animales , Antibacterianos/farmacología , Anticuerpos/genética , Anticuerpos/metabolismo , Linfocitos B/metabolismo , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Memoria Inmunológica/inmunología , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Microbiota/genética , Microbiota/fisiología , Mutación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , ARN Ribosómico 16S/genética , Simbiosis/efectos de los fármacos , Simbiosis/inmunología , Adulto JovenRESUMEN
Although vaccines confer protection against influenza A viruses, antiviral treatment becomes the first line of defense during pandemics because there is insufficient time to produce vaccines. Current antiviral drugs are susceptible to drug resistance, and developing new antivirals is essential. We studied host defense peptides from the skin of the South Indian frog and demonstrated that one of these, which we named "urumin," is virucidal for H1 hemagglutinin-bearing human influenza A viruses. This peptide specifically targeted the conserved stalk region of H1 hemagglutinin and was effective against drug-resistant H1 influenza viruses. Using electron microscopy, we showed that this peptide physically destroyed influenza virions. It also protected naive mice from lethal influenza infection. Urumin represents a unique class of anti-influenza virucide that specifically targets the hemagglutinin stalk region, similar to targeting of antibodies induced by universal influenza vaccines. Urumin therefore has the potential to contribute to first-line anti-viral treatments during influenza outbreaks.
Asunto(s)
Proteínas Anfibias/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/prevención & control , Infecciones por Orthomyxoviridae/prevención & control , Péptidos/farmacología , Secuencia de Aminoácidos , Proteínas Anfibias/inmunología , Animales , Antivirales/inmunología , Antivirales/farmacología , Perros , Relación Dosis-Respuesta a Droga , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Virus de la Influenza A/inmunología , Virus de la Influenza A/metabolismo , Gripe Humana/inmunología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Péptidos/inmunología , Ranidae/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Virión/efectos de los fármacos , Virión/inmunología , Virión/metabolismoRESUMEN
Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.
Asunto(s)
Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Antígeno CTLA-4/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Antígeno CTLA-4/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , VIH-1/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Hibridación in Situ , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Macaca mulatta , Microscopía Confocal , Receptor de Muerte Celular Programada 1/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/virología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/virologíaRESUMEN
A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and has been rapidly spreading around the globe1,2. SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome3 and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.
Asunto(s)
Betacoronavirus/efectos de los fármacos , Betacoronavirus/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/metabolismo , Terapia Molecular Dirigida , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/metabolismo , Proteómica , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Células CACO-2 , Carbono/metabolismo , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Técnicas In Vitro , Cinética , Pandemias , Neumonía Viral/genética , Neumonía Viral/virología , Biosíntesis de Proteínas/efectos de los fármacos , Proteoma/metabolismo , Proteostasis , Empalme del ARN , SARS-CoV-2 , Factores de Tiempo , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
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Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/metabolismo , Reposicionamiento de Medicamentos , Terapia Molecular Dirigida , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/metabolismo , Mapas de Interacción de Proteínas , Proteínas Virales/metabolismo , Animales , Antivirales/clasificación , Antivirales/farmacología , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidad , COVID-19 , Chlorocebus aethiops , Clonación Molecular , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad Innata , Espectrometría de Masas , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Dominios Proteicos , Mapeo de Interacción de Proteínas , Receptores sigma/metabolismo , SARS-CoV-2 , Proteínas Ligasas SKP Cullina F-box/metabolismo , Células Vero , Proteínas Virales/genética , Tratamiento Farmacológico de COVID-19RESUMEN
Vibrio cholerae is the causative agent of cholera, a potentially lethal enteric bacterial infection1. Cholera toxin (CTX), a protein complex that is secreted by V. cholerae, is required for V. cholerae to cause severe disease. CTX is also thought to promote transmission of the organism, as infected individuals shed many litres of diarrhoeal fluid that typically contains in excess of 1011 organisms per litre. How the pathogen is able to reach such high concentrations in the intestine during infection remains poorly understood. Here we show that CTX increases pathogen growth and induces a distinct V. cholerae transcriptomic signature that is indicative of an iron-depleted gut niche. During infection, bacterial pathogens need to acquire iron, which is an essential nutrient for growth2. Most iron in the mammalian host is found in a chelated form within the porphyrin structure of haem, and the ability to use haem as a source of iron is genetically encoded by V. cholerae3. We show that the genes that enable V. cholerae to obtain iron via haem and vibriobactin confer a growth advantage to the pathogen only when CTX is produced. Furthermore, we found that CTX-induced congestion of capillaries in the terminal ileum correlated with an increased bioavailability of luminal haem. CTX-induced disease in the ileum also led to increased concentrations of long-chain fatty acids and L-lactate metabolites in the lumen, as well as the upregulation of V. cholerae genes that encode enzymes of the tricarboxylic acid (TCA) cycle that contain iron-sulfur clusters. Genetic analysis of V. cholerae suggested that pathogen growth was dependent on the uptake of haem and long-chain fatty acids during infection, but only in a strain capable of producing CTX in vivo. We conclude that CTX-induced disease creates an iron-depleted metabolic niche in the gut, which selectively promotes the growth of V. cholerae through the acquisition of host-derived haem and fatty acids.
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Toxina del Cólera/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/metabolismo , Animales , Cólera/metabolismo , Cólera/microbiología , Ciclo del Ácido Cítrico/efectos de los fármacos , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Hemo/metabolismo , Ácido Láctico/metabolismo , Ratones , Conejos , Análisis de Secuencia de ARN , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Vibrio cholerae/genéticaRESUMEN
SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.
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Regiones no Traducidas 5'/genética , Evasión Inmune/genética , Biosíntesis de Proteínas , SARS-CoV-2/genética , Proteínas no Estructurales Virales/genética , Animales , Secuencia de Bases , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Evasión Inmune/efectos de los fármacos , Ratones Transgénicos , Modelos Biológicos , Oligonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.
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Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , Interacciones Huésped-Patógeno , Terapia Molecular Dirigida , Procesamiento Proteico-Postraduccional , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/metabolismo , COVID-19/virología , Células CACO-2 , Exorribonucleasas/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Sirtuinas/metabolismo , Succinatos/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.
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Autofagia , Interacciones Huésped-Patógeno , Integrina alfa5beta1 , Receptores Purinérgicos P2Y2 , Serratia , Toxinas Biológicas , Animales , Cricetinae , Adenosina Trifosfato/metabolismo , Autofagia/efectos de los fármacos , Células CHO , Cricetulus , Exocitosis/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Serratia/química , Serratia/efectos de los fármacos , Serratia/fisiología , Toxinas Biológicas/farmacología , HumanosRESUMEN
The development of physiological models that reproduce SARS-CoV-2 infection in primary human cells will be instrumental to identify host-pathogen interactions and potential therapeutics. Here, using cell suspensions directly from primary human lung tissues (HLT), we have developed a rapid platform for the identification of viral targets and the expression of viral entry factors, as well as for the screening of viral entry inhibitors and anti-inflammatory compounds. The direct use of HLT cells, without long-term cell culture and in vitro differentiation approaches, preserves main immune and structural cell populations, including the most susceptible cell targets for SARS-CoV-2; alveolar type II (AT-II) cells, while maintaining the expression of proteins involved in viral infection, such as ACE2, TMPRSS2, CD147 and AXL. Further, antiviral testing of 39 drug candidates reveals a highly reproducible method, suitable for different SARS-CoV-2 variants, and provides the identification of new compounds missed by conventional systems, such as VeroE6. Using this method, we also show that interferons do not modulate ACE2 expression, and that stimulation of local inflammatory responses can be modulated by different compounds with antiviral activity. Overall, we present a relevant and rapid method for the study of SARS-CoV-2.