Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
PLoS Pathog ; 19(7): e1011351, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37410700

RESUMO

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.


Assuntos
COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Animais , Humanos , Camundongos , Enzima de Conversão de Angiotensina 2/metabolismo , Dipeptidil Peptidase 4/metabolismo , Histona Desmetilases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo
2.
Proc Natl Acad Sci U S A ; 115(36): E8421-E8429, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127032

RESUMO

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.


Assuntos
Exocitose/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Proteínas Munc18 , Peptídeos , Proteínas SNARE , Animais , Células COS , Chlorocebus aethiops , Cinética , Camundongos , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ratos , Proteínas SNARE/química , Proteínas SNARE/metabolismo
3.
Sci Immunol ; 9(93): eadi7038, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38517952

RESUMO

The persistent murine norovirus strain MNVCR6 is a model for human norovirus and enteric viral persistence. MNVCR6 causes chronic infection by directly infecting intestinal tuft cells, rare chemosensory epithelial cells. Although MNVCR6 induces functional MNV-specific CD8+ T cells, these lymphocytes fail to clear infection. To examine how tuft cells promote immune escape, we interrogated tuft cell interactions with CD8+ T cells by adoptively transferring JEDI (just EGFP death inducing) CD8+ T cells into Gfi1b-GFP tuft cell reporter mice. Unexpectedly, some intestinal tuft cells partially resisted JEDI CD8+ T cell-mediated killing-unlike Lgr5+ intestinal stem cells and extraintestinal tuft cells-despite seemingly normal antigen presentation. When targeting intestinal tuft cells, JEDI CD8+ T cells predominantly adopted a T resident memory phenotype with decreased effector and cytotoxic capacity, enabling tuft cell survival. JEDI CD8+ T cells neither cleared nor prevented MNVCR6 infection in the colon, the site of viral persistence, despite targeting a virus-independent antigen. Ultimately, we show that intestinal tuft cells are relatively resistant to CD8+ T cells independent of norovirus infection, representing an immune-privileged niche that can be leveraged by enteric microbes.


Assuntos
Linfócitos T CD8-Positivos , Norovirus , Camundongos , Humanos , Animais , Células em Tufo , Norovirus/fisiologia , Privilégio Imunológico , Intestinos
4.
Nat Microbiol ; 9(8): 2038-2050, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39075235

RESUMO

Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.


Assuntos
COVID-19 , Quirópteros , Imunidade Inata , SARS-CoV-2 , Replicação Viral , Animais , Humanos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Quirópteros/virologia , Quirópteros/imunologia , COVID-19/transmissão , COVID-19/virologia , COVID-19/imunologia , Camundongos , Cricetinae , Evasão da Resposta Imune , Células Epiteliais/virologia , Células Epiteliais/imunologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Coronavirus/imunologia , Coronavirus/genética , Coronavirus/classificação , Coronavirus/fisiologia , Coronavirus/patogenicidade , Linhagem Celular , Feminino
5.
bioRxiv ; 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37693555

RESUMO

COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.

6.
Cell Rep ; 42(12): 113478, 2023 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-37991919

RESUMO

Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.


Assuntos
COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , SARS-CoV-2 , Cromatina , Proteômica , Senescência Celular , Células Gigantes , Proteína Supressora de Tumor p53/genética
7.
Nat Genet ; 55(3): 471-483, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36894709

RESUMO

Identification of host determinants of coronavirus infection informs mechanisms of viral pathogenesis and can provide new drug targets. Here we demonstrate that mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complexes, specifically canonical BRG1/BRM-associated factor (cBAF) complexes, promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and represent host-directed therapeutic targets. The catalytic activity of SMARCA4 is required for mSWI/SNF-driven chromatin accessibility at the ACE2 locus, ACE2 expression and virus susceptibility. The transcription factors HNF1A/B interact with and recruit mSWI/SNF complexes to ACE2 enhancers, which contain high HNF1A motif density. Notably, small-molecule mSWI/SNF ATPase inhibitors or degraders abrogate angiotensin-converting enzyme 2 (ACE2) expression and confer resistance to SARS-CoV-2 variants and a remdesivir-resistant virus in three cell lines and three primary human cell types, including airway epithelial cells, by up to 5 logs. These data highlight the role of mSWI/SNF complex activities in conferring SARS-CoV-2 susceptibility and identify a potential class of broad-acting antivirals to combat emerging coronaviruses and drug-resistant variants.


Assuntos
COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2/genética , Cromatina , COVID-19/genética , DNA Helicases/genética , Proteínas Nucleares/genética , SARS-CoV-2 , Fatores de Transcrição/genética
8.
J Exp Med ; 219(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34757384

RESUMO

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral infection in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I)-dependent manner. SLR14 demonstrated remarkable prophylactic protective capacity against lethal SARS-CoV-2 infection and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity in the absence of the adaptive immune system. In the context of infection with variants of concern (VOCs), SLR14 conferred broad protection against emerging VOCs. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and treatment of chronically infected immunosuppressed patients.


Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/metabolismo , Modelos Animais de Doenças , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
9.
Sci Immunol ; 7(68): eabl5652, 2022 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-34914544

RESUMO

T follicular helper (TFH) cells are the conventional drivers of protective, germinal center (GC)­based antiviral antibody responses. However, loss of TFH cells and GCs has been observed in patients with severe COVID-19. As T cell­B cell interactions and immunoglobulin class switching still occur in these patients, noncanonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both TFH-dependent and -independent antibodies were induced against SARS-CoV-2 infection, SARS-CoV-2 vaccination, and influenza A virus infection. Although TFH-independent antibodies to SARS-CoV-2 had evidence of reduced somatic hypermutation, they were still high affinity, durable, and reactive against diverse spike-derived epitopes and were capable of neutralizing both homologous SARS-CoV-2 and the B.1.351 (beta) variant of concern. We found by epitope mapping and B cell receptor sequencing that TFH cells focused the B cell response, and therefore, in the absence of TFH cells, a more diverse clonal repertoire was maintained. These data support an alternative pathway for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GC-derived antibodies that might compensate for GCs damaged by viral inflammation.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Células T Auxiliares Foliculares/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , Centro Germinativo/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Auxiliares-Indutores
10.
bioRxiv ; 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34159330

RESUMO

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral replication in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I) dependent manner. SLR14 demonstrated remarkable protective capacity against lethal SARS-CoV-2 infection when used prophylactically and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity by inducing IFN-I responses in the absence of the adaptive immune system. In the context of infection with variants of concern (VOC), SLR14 conferred broad protection and uncovered an IFN-I resistance gradient across emerging VOC. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and for treatment of chronically infected immunosuppressed patients.

11.
Sci Adv ; 6(48)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33246952

RESUMO

Major histocompatibility complex (MHC)-unrestricted cytotoxic lymphocytes (CLs) such as natural killer (NK) cells can detect and destroy tumor and virus-infected cells resistant to T cell-mediated killing. Here, we performed genome-wide genetic screens to identify tumor-intrinsic genes regulating killing by MHC-unrestricted CLs. A group of genes identified in our screens encode enzymes for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is not involved in tumor response to T cell-mediated cytotoxicity. Another gene identified in the screens was PBRM1, which encodes a subunit of the PBAF form of the SWI/SNF chromatin-remodeling complex. PBRM1 mutations in tumor cells cause resistance to MHC-unrestricted killing, in contrast to their sensitizing effects on T cell-mediated killing. PBRM1 and the GPI biosynthetic pathway regulate the ligands of NK cell receptors in tumor cells and promote cytolytic granule secretion in CLs. The regulators identified in this work represent potential targets for cancer immunotherapy.


Assuntos
Citotoxicidade Imunológica , Neoplasias , Vias Biossintéticas , Proteínas de Ligação a DNA/genética , Glicosilfosfatidilinositóis , Antígenos de Histocompatibilidade , Humanos , Células Matadoras Naturais , Complexo Principal de Histocompatibilidade , Neoplasias/genética , Fatores de Transcrição/genética
12.
Curr Protoc Cell Biol ; 82(1): e68, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30265447

RESUMO

About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.


Assuntos
Transporte Biológico/genética , Membrana Celular/metabolismo , Citometria de Fluxo , Testes Genéticos , Animais , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
13.
Dev Cell ; 50(4): 436-446.e5, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31353312

RESUMO

Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Endocitose/genética , Sequência de Aminoácidos/genética , Membrana Celular/genética , Clatrina/genética , Humanos , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/patologia , Ligação Proteica/genética , Transporte Proteico/genética , Proteólise
14.
Cell Rep ; 11(11): 1727-36, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26074080

RESUMO

Glycophosphatidylinositol-anchored proteins (GPI-APs) play essential roles in physiology, but their biogenesis and trafficking have not been systematically characterized. Here, we took advantage of the recently available haploid genetics approach to dissect GPI-AP pathways in human cells using prion protein (PrP) and CD59 as model molecules. Our screens recovered a large number of common and unexpectedly specialized factors in the GPI-AP pathways. PIGN, PGAP2, and PIGF, which encode GPI anchor-modifying enzymes, were selectively isolated in the CD59 screen, suggesting that GPI anchor composition significantly influences the biogenesis of GPI-APs in a substrate-dependent manner. SEC62 and SEC63, which encode components of the ER-targeting machinery, were selectively recovered in the PrP screen, indicating that they do not constitute a universal route for the biogenesis of mammalian GPI-APs. Together, these comparative haploid genetic screens demonstrate that, despite their similarity in overall architecture and subcellular localization, GPI-APs follow markedly distinct biosynthetic and trafficking pathways.


Assuntos
Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/genética , Haploidia , Antígenos CD59/genética , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Glicosilfosfatidilinositóis/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Príons/metabolismo , Transporte Proteico , Proteínas de Ligação a RNA
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa