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1.
Cell ; 184(1): 92-105.e16, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33147445

RESUMO

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.


Assuntos
COVID-19/genética , COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Vias Biossintéticas , COVID-19/metabolismo , Colesterol/biossíntese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endossomos/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes/métodos , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferência de RNA , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Carga Viral/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
2.
Cell ; 181(7): 1502-1517.e23, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32559462

RESUMO

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.


Assuntos
Capuzes de RNA/genética , Infecções por Vírus de RNA/genética , Proteínas Recombinantes de Fusão/genética , Regiões 5' não Traduzidas/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Cães , Humanos , Vírus da Influenza A/metabolismo , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Fases de Leitura Aberta/genética , Capuzes de RNA/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
3.
Cell ; 172(4): 811-824.e14, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29395325

RESUMO

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.


Assuntos
Adenosina Desaminase/metabolismo , Elementos Alu , Doenças Autoimunes do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Biossíntese de Proteínas , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Morte Celular/genética , Morte Celular/imunologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/patologia , RNA Polimerase II/genética , RNA Polimerase II/imunologia , RNA Polimerase II/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , eIF-2 Quinase/genética , eIF-2 Quinase/imunologia , eIF-2 Quinase/metabolismo
4.
Cell ; 172(3): 423-438.e25, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29249360

RESUMO

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.


Assuntos
Imunidade Inata , Células-Tronco Pluripotentes/imunologia , Viroses/imunologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/virologia , Especificidade da Espécie
5.
Cell ; 172(5): 952-965.e18, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29474921

RESUMO

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.


Assuntos
Encefalopatias Metabólicas Congênitas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , RNA/química , RNA/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Encefalopatias Metabólicas Congênitas/patologia , Tronco Encefálico/patologia , Encefalite Viral/genética , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1 , Humanos , Interferons/metabolismo , Íntrons/genética , Masculino , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Linhagem , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Receptor 3 Toll-Like/metabolismo , Replicação Viral
6.
Mol Cell ; 83(23): 4255-4271.e9, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37995687

RESUMO

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.


Assuntos
Retrovirus Endógenos , Retrovirus Endógenos/genética , RNA Nuclear , Epigênese Genética , Heterocromatina , Expressão Gênica
7.
Immunity ; 53(3): 672-684.e11, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32750333

RESUMO

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.


Assuntos
Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/patologia , Janus Quinase 1/genética , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/patologia , Adolescente , COVID-19/mortalidade , Domínio Catalítico/genética , Linhagem Celular , Citocinas/metabolismo , Feminino , Mutação com Ganho de Função/genética , Genótipo , Células HEK293 , Doenças Hereditárias Autoinflamatórias/tratamento farmacológico , Humanos , Janus Quinase 1/antagonistas & inibidores , Mosaicismo , Piperidinas/uso terapêutico , Medicina de Precisão/métodos , Pirimidinas/uso terapêutico , Transdução de Sinais/imunologia , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico
8.
Nature ; 615(7951): 305-314, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36813963

RESUMO

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata1,2. Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c+TbethighCD21low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition.


Assuntos
Autoimunidade , Linfócitos T CD4-Positivos , Citocinas , Síndrome de Down , Humanos , Autoanticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/análise , Citocinas/imunologia , Suscetibilidade a Doenças , Síndrome de Down/imunologia , Síndrome de Down/fisiopatologia , Interleucina-6/imunologia , Receptores de Complemento 3d
9.
Mol Cell ; 74(6): 1264-1277.e7, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31130363

RESUMO

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.


Assuntos
Ciclo Celular/genética , Ciclinas/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F3/genética , Proteínas Ligases SKP Culina F-Box/genética , Transcrição Gênica , Linhagem Celular Tumoral , Ciclinas/metabolismo , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F3/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Aptidão Genética , Células HEK293 , Células HeLa , Humanos , Mutação , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteólise , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Ubiquitinação
10.
Annu Rev Genet ; 51: 241-263, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28853921

RESUMO

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.


Assuntos
Imunidade Adaptativa , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Viroses/genética , Citocinas/genética , Citocinas/imunologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/imunologia , Genética Humana , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Receptores KIR/genética , Receptores KIR/imunologia , Receptores Virais/genética , Receptores Virais/imunologia , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/imunologia , Viroses/imunologia , Viroses/patologia , Viroses/virologia
11.
Nat Immunol ; 14(7): 749-55, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708250

RESUMO

Beyond its well-characterized functions in antibody diversification, the cytidine deaminase AID can catalyze off-target DNA damage and has been hypothesized to edit RNA and mediate DNA demethylation. To comprehensively examine the effects of AID on the transcriptome and the pattern of DNA methylation ('methylome'), we analyzed AID-deficient (Aicda(-/-)), wild-type and AID-overexpressing activated B cells by high-throughput RNA sequencing (RNA-Seq) and reduced-representation bisulfite sequencing (RRBS). These analyses confirmed the known role of AID in immunoglobulin isotype switching and also demonstrated few other effects of AID on gene expression. Additionally, we detected no evidence of AID-dependent editing of mRNA or microRNA. Finally, the RRBS data did not support the proposed role for AID in regulating DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification seems to be the sole physiological function of AID in activated B cells.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/imunologia , Isotipos de Imunoglobulinas/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Transcriptoma/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/química , MicroRNAs/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Sequência de DNA
12.
PLoS Pathog ; 18(4): e1010464, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35421191

RESUMO

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.


Assuntos
2',5'-Oligoadenilato Sintetase , COVID-19 , Interferons , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Antivirais/farmacologia , COVID-19/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Interferons/metabolismo , SARS-CoV-2/genética
13.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723056

RESUMO

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.


Assuntos
Adenosina Desaminase/genética , Regulação da Expressão Gênica , Isoformas de Proteínas/genética , Edição de RNA , Proteínas de Ligação a RNA/genética , Doenças Autoimunes do Sistema Nervoso/genética , Suscetibilidade a Doenças , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Humanos , Malformações do Sistema Nervoso/genética , Transtornos da Pigmentação/congênito , Transtornos da Pigmentação/genética
14.
Respir Res ; 24(1): 213, 2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37635251

RESUMO

BACKGROUND: The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized. METHODS: BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens. RESULTS: We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. CONCLUSIONS: Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.


Assuntos
Células Epiteliais , Interferons , Humanos , Epitélio , Diferenciação Celular , Expressão Gênica
15.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33097660

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.


Assuntos
COVID-19/metabolismo , Interferons/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Transdução de Sinais , Células Vero
16.
J Virol ; 95(23): e0125721, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34523966

RESUMO

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.


Assuntos
COVID-19/metabolismo , Citocinas/metabolismo , Interferon Tipo I/metabolismo , SARS-CoV-2 , Fator de Transcrição RelA/metabolismo , Transcriptoma , Replicação Viral , Células A549 , Animais , COVID-19/virologia , Chlorocebus aethiops , Epigenômica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Interações entre Hospedeiro e Microrganismos , Humanos , Transdução de Sinais , Análise de Célula Única , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fatores de Transcrição/metabolismo , Células Vero
17.
PLoS Pathog ; 16(7): e1008677, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32649726

RESUMO

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.


Assuntos
Medula Óssea/virologia , Infecções por Flavivirus/veterinária , Hepatite Viral Animal/virologia , Doenças dos Cavalos/virologia , Animais , Flaviviridae , Infecções por Flavivirus/virologia , Cavalos
18.
Hepatology ; 74(3): 1148-1163, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33713356

RESUMO

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.


Assuntos
Regulação da Expressão Gênica , Hepacivirus/imunologia , Hepatite Viral Animal/imunologia , Fígado/imunologia , MicroRNAs/imunologia , Linfócitos T/imunologia , Animais , Progressão da Doença , Hepacivirus/metabolismo , Hepatite Viral Animal/genética , Cavalos , Fígado/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Transcriptoma
19.
BMC Biol ; 19(1): 13, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33482825

RESUMO

BACKGROUND: Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. RESULTS: Here, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1- lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. CONCLUSIONS: Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.


Assuntos
Cavalos/sangue , Leucócitos Mononucleares/classificação , Animais , Linfócitos B/classificação , Leucócitos Mononucleares/metabolismo , Análise de Sequência de RNA/veterinária , Análise de Célula Única/veterinária
20.
PLoS Pathog ; 15(11): e1007634, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682641

RESUMO

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.


Assuntos
Antivirais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vírus da Influenza A/imunologia , Interferon Tipo I/farmacologia , Proteínas Nucleares/metabolismo , Infecções por Orthomyxoviridae/imunologia , Fatores de Transcrição/metabolismo , Replicação Viral/imunologia , Células A549 , Animais , Feminino , Humanos , Imunidade Inata , Vírus da Influenza A/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Fosforilação , Fator de Transcrição STAT1 , Transdução de Sinais , Fatores de Transcrição/genética , Replicação Viral/efeitos dos fármacos
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