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1.
Cell ; 185(3): 493-512.e25, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-35032429

RESUMEN

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.


Asunto(s)
COVID-19/inmunología , COVID-19/patología , Activación de Complemento , Proteoma , SARS-CoV-2/inmunología , Linfocitos T Citotóxicos/inmunología , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/virología , Factores Quimiotácticos/metabolismo , Citotoxicidad Inmunológica , Células Endoteliales/virología , Femenino , Humanos , Activación de Linfocitos , Masculino , Microvasos/virología , Persona de Mediana Edad , Monocitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Análisis de la Célula Individual , Adulto Joven
2.
Cell ; 184(26): 6243-6261.e27, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34914922

RESUMEN

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.


Asunto(s)
COVID-19/patología , COVID-19/virología , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/virología , Macrófagos/patología , Macrófagos/virología , SARS-CoV-2/fisiología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , COVID-19/diagnóstico por imagen , Comunicación Celular , Estudios de Cohortes , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/diagnóstico por imagen , Fibrosis Pulmonar Idiopática/genética , Células Madre Mesenquimatosas/patología , Fenotipo , Proteoma/metabolismo , Receptores de Superficie Celular/metabolismo , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/virología , Tomografía Computarizada por Rayos X , Transcripción Genética
3.
Cell ; 182(6): 1419-1440.e23, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32810438

RESUMEN

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.


Asunto(s)
Infecciones por Coronavirus/inmunología , Células Mieloides/inmunología , Mielopoyesis , Neumonía Viral/inmunología , Adulto , Anciano , Antígenos CD11/genética , Antígenos CD11/metabolismo , COVID-19 , Células Cultivadas , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/patología , Femenino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Células Mieloides/citología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/patología , Proteoma/genética , Proteoma/metabolismo , Proteómica , Análisis de la Célula Individual
4.
Cell ; 178(1): 242-260.e29, 2019 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-31155234

RESUMEN

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.


Asunto(s)
Miocardio/metabolismo , Biosíntesis de Proteínas , Adolescente , Adulto , Anciano , Animales , Codón/genética , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Sistemas de Lectura Abierta/genética , ARN Circular/genética , ARN Circular/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ribosomas/genética , Ribosomas/metabolismo , Adulto Joven
5.
Nature ; 599(7884): 283-289, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34517409

RESUMEN

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19/patología , COVID-19/virología , Senescencia Celular/efectos de los fármacos , Terapia Molecular Dirigida , SARS-CoV-2/patogenicidad , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Animales , COVID-19/complicaciones , Línea Celular , Cricetinae , Dasatinib/farmacología , Dasatinib/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Quercetina/farmacología , Quercetina/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Trombosis/complicaciones , Trombosis/inmunología , Trombosis/metabolismo
6.
PLoS Pathog ; 19(11): e1011759, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37967063

RESUMEN

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.


Asunto(s)
Hepacivirus , Hepatitis C , Humanos , Hepacivirus/fisiología , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Internalización del Virus , Proteínas Portadoras , Receptores ErbB/metabolismo , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo
7.
Mol Cell ; 66(1): 9-21.e7, 2017 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-28344080

RESUMEN

Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.


Asunto(s)
Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/metabolismo , Proteínas Nucleares/biosíntesis , Biosíntesis de Proteínas , ARN/metabolismo , Ribosomas/metabolismo , Animales , Línea Celular , Codón Iniciador , Codón de Terminación , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Factores de Transcripción Forkhead/metabolismo , Genotipo , Cabeza , Espectrometría de Masas , Ratones , Mutación , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Estado Nutricional , Fenotipo , ARN/química , ARN/genética , Caperuzas de ARN/química , Caperuzas de ARN/genética , ARN Circular , Ratas , Ribosomas/química , Ribosomas/genética , Inanición/genética , Inanición/metabolismo , Relación Estructura-Actividad , Transfección
8.
J Virol ; 97(6): e0040023, 2023 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-37289084

RESUMEN

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.


Asunto(s)
Infecciones por Citomegalovirus , Elementos de Facilitación Genéticos , Factor 3 Regulador del Interferón , Interferón Tipo I , Proteínas de la Matriz Viral , Animales , Humanos , Ratones , Infecciones por Citomegalovirus/genética , ADN/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Muromegalovirus/genética , Muromegalovirus/metabolismo , Proteínas de la Matriz Viral/metabolismo
9.
PLoS Pathog ; 18(1): e1010193, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34982803

RESUMEN

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.


Asunto(s)
Citomegalovirus/genética , Pan troglodytes/virología , Animales , Infecciones por Citomegalovirus/virología , Modelos Animales de Enfermedad , Genoma Viral , Humanos
10.
Mol Ther ; 30(5): 1952-1965, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35339689

RESUMEN

For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Anticuerpos Antivirales , Antivirales , Cricetinae , Dexametasona/farmacología , SARS-CoV-2 , Transcriptoma
11.
Nucleic Acids Res ; 49(9): 5336-5350, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-33905506

RESUMEN

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.


Asunto(s)
Regiones no Traducidas 5' , ARN Helicasas DEAD-box/fisiología , Biosíntesis de Proteínas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Humanos , Mutación , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , ARN Interferente Pequeño
12.
Int J Mol Sci ; 24(24)2023 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-38139252

RESUMEN

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.


Asunto(s)
Ciclinas , Infecciones por Herpesviridae , Humanos , Ciclinas/metabolismo , Citomegalovirus/genética , Ciclina H/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Fosforilación
13.
J Proteome Res ; 21(6): 1575-1587, 2022 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-35608653

RESUMEN

Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites is not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge. A recent large-scale study systematically identified in vitro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic data sets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic data sets.


Asunto(s)
COVID-19 , Fosfoproteínas , Humanos , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , SARS-CoV-2
14.
Eur Respir J ; 60(6)2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-35728978

RESUMEN

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.


Asunto(s)
COVID-19 , Gripe Humana , Adulto , Humanos , Enzima Convertidora de Angiotensina 2 , Pulmón/patología , Macrófagos Alveolares/metabolismo , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Tropismo Viral
15.
Int J Mol Sci ; 23(19)2022 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-36233116

RESUMEN

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.


Asunto(s)
Ciclina H , Citomegalovirus , Proteínas Virales , Aminoácidos/metabolismo , Ciclina H/genética , Ciclina H/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Citomegalovirus/fisiología , Marcadores Genéticos , Humanos , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Virales/genética , Replicación Viral/genética
16.
Mol Cell ; 46(5): 674-90, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22681889

RESUMEN

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.


Asunto(s)
Proteómica/métodos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Línea Celular , Humanos , Espectrometría de Masas , Proteínas de Unión al ARN/química , Análisis de Secuencia de ARN
18.
Nat Methods ; 13(2): 165-70, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26657557

RESUMEN

RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.


Asunto(s)
Ribosomas/metabolismo , Células HEK293 , Humanos , Sistemas de Lectura Abierta , Biosíntesis de Proteínas/genética , Ribosomas/genética , Transcriptoma
19.
Mol Syst Biol ; 13(5): 928, 2017 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-28468958

RESUMEN

The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals. We obtained both protein phosphorylation and gene expression time course data from HEK293 cells carrying an inducible construct of the proto-oncogene RAF By mathematical modelling, we identified a new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function. We find that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis shows that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, our findings suggest that ILGs function as a gene expression module to decode ERK signal duration.


Asunto(s)
Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , ARN Mensajero/metabolismo , Animales , Simulación por Computador , Secuencia Rica en GC , Células HEK293 , Semivida , Humanos , Células MCF-7 , Modelos Teóricos , Familia de Multigenes , Células PC12 , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Ratas , Transducción de Señal/genética , Quinasas raf/genética
20.
BMC Bioinformatics ; 18(1): 7, 2017 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-28049429

RESUMEN

BACKGROUND: RNA editing is a co-transcriptional modification that increases the molecular diversity, alters secondary structure and protein coding sequences by changing the sequence of transcripts. The most common RNA editing modification is the single base substitution (A→I) that is catalyzed by the members of the Adenosine deaminases that act on RNA (ADAR) family. Typically, editing sites are identified as RNA-DNA-differences (RDDs) in a comparison of genome and transcriptome data from next-generation sequencing experiments. However, a method for robust detection of site-specific editing events from replicate RNA-seq data has not been published so far. Even more surprising, condition-specific editing events, which would show up as differences in RNA-RNA comparisons (RRDs) and depend on particular cellular states, are rarely discussed in the literature. RESULTS: We present JACUSA, a versatile one-stop solution to detect single nucleotide variant positions from comparing RNA-DNA and/or RNA-RNA sequencing samples. The performance of JACUSA has been carefully evaluated and compared to other variant callers in an in silico benchmark. JACUSA outperforms other algorithms in terms of the F measure, which combines precision and recall, in all benchmark scenarios. This performance margin is highest for the RNA-RNA comparison scenario. We further validated JACUSA's performance by testing its ability to detect A→I events using sequencing data from a human cell culture experiment and publicly available RNA-seq data from Drosophila melanogaster heads. To this end, we performed whole genome and RNA sequencing of HEK-293 cells on samples with lowered activity of candidate RNA editing enzymes. JACUSA has a higher recall and comparable precision for detecting true editing sites in RDD comparisons of HEK-293 data. Intriguingly, JACUSA captures most A→I events from RRD comparisons of RNA sequencing data derived from Drosophila and HEK-293 data sets. CONCLUSION: Our software JACUSA detects single nucleotide variants by comparing data from next-generation sequencing experiments (RNA-DNA or RNA-RNA). In practice, JACUSA shows higher recall and comparable precision in detecting A→I sites from RNA-DNA comparisons, while showing higher precision and recall in RNA-RNA comparisons.


Asunto(s)
Edición de ARN , ARN/metabolismo , Programas Informáticos , Animales , Drosophila melanogaster/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Polimorfismo de Nucleótido Simple , ARN/química , Interferencia de ARN , Análisis de Secuencia de ARN , Transcriptoma
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