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1.
J Exp Med ; 216(9): 2057-2070, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31270247

RESUMO

Vaccination against measles, mumps, and rubella (MMR) and yellow fever (YF) with live attenuated viruses can rarely cause life-threatening disease. Severe illness by MMR vaccines can be caused by inborn errors of type I and/or III interferon (IFN) immunity (mutations in IFNAR2, STAT1, or STAT2). Adverse reactions to the YF vaccine have remained unexplained. We report two otherwise healthy patients, a 9-yr-old boy in Iran with severe measles vaccine disease at 1 yr and a 14-yr-old girl in Brazil with viscerotropic disease caused by the YF vaccine at 12 yr. The Iranian patient is homozygous and the Brazilian patient compound heterozygous for loss-of-function IFNAR1 variations. Patient-derived fibroblasts are susceptible to viruses, including the YF and measles virus vaccine strains, in the absence or presence of exogenous type I IFN. The patients' fibroblast phenotypes are rescued with WT IFNAR1 Autosomal recessive, complete IFNAR1 deficiency can result in life-threatening complications of vaccination with live attenuated measles and YF viruses in previously healthy individuals.

2.
Nat Chem Biol ; 15(3): 259-268, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30643282

RESUMO

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Vesículas Transportadoras/fisiologia , Células A549 , Animais , Antígenos de Diferenciação/metabolismo , Antivirais , Endossomos/fisiologia , Células HeLa , Humanos , Lisossomos/fisiologia , Imagem Óptica/métodos , Transporte Proteico , Vírion/patogenicidade , Internalização do Vírus
3.
Cell Rep ; 25(4): 833-840.e3, 2018 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-30355490

RESUMO

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85ß, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

4.
Cell ; 172(3): 423-438.e25, 2018 Jan 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.

5.
Sci Rep ; 7(1): 16616, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-29192196

RESUMO

The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the hepatitis B virus (HBV) receptor enabled researchers to create hepatoma cell lines susceptible to HBV infection. Infection in current systems, however, is inefficient and virus fails to spread. Infection efficiency is enhanced by treating cells with polyethylene glycol 8000 (PEG) during infection. However, this alone does not promote virus spread. Here we show that maintaining PEG in culture medium increases the rate of infection by at least one order of magnitude, and, most importantly, promotes virus spread. To demonstrate the utility of this system, we show that two interferon-stimulated genes (ISGs), ISG20 and tetherin, restrict HBV spread in NTCP-expressing hepatoma cells. Thus, this protocol can be easily applied to existing cell culture systems to study the complete HBV life cycle, including virus spread.

6.
Immunity ; 46(3): 379-392, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28329704

RESUMO

Type III interferons (IFN-λs) signal through a heterodimeric receptor complex composed of the IFN-λR1 subunit, specific for IFN-λs, and interleukin-10Rß (IL-10Rß), which is shared by multiple cytokines in the IL-10 superfamily. Low affinity of IL-10Rß for cytokines has impeded efforts aimed at crystallizing cytokine-receptor complexes. We used yeast surface display to engineer a higher-affinity IFN-λ variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL-10Rß uses a network of tyrosine residues as hydrophobic anchor points to engage IL-10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross-reactive but cytokine-specific receptor. H11 elicited increased anti-proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher-affinity type I IFNs did not increase antiviral potency over wild-type type I IFNs. Our findings provide insight into cytokine recognition by the IL-10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential.


Assuntos
Interferons/imunologia , Receptores de Interferon/imunologia , Receptores de Interleucina-10/imunologia , Animais , Linhagem Celular , Cristalografia por Raios X , Citometria de Fluxo , Humanos , Camundongos , Reação em Cadeia da Polimerase , Ressonância de Plasmônio de Superfície
7.
J Exp Med ; 213(13): 2931-2947, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27899441

RESUMO

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2-/- mice after peripheral inoculation. Irf2-/- mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2-/- mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient µMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2-/- mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections.


Assuntos
Infecções por Alphavirus/imunologia , Linfócitos B/imunologia , Encefalopatias/imunologia , Movimento Celular/imunologia , Fator Regulador 2 de Interferon/imunologia , Vírus Sindbis/imunologia , Infecções por Alphavirus/genética , Infecções por Alphavirus/patologia , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linfócitos B/patologia , Encefalopatias/genética , Encefalopatias/patologia , Encefalopatias/virologia , Movimento Celular/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Fator Regulador 2 de Interferon/genética , Camundongos , Camundongos Knockout
8.
Virology ; 494: 236-47, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27128351

RESUMO

Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have not been studied at the single-cell level, leaving a gap in our understanding of antiviral kinetics and host-virus interactions. Here, we demonstrate quantitative imaging of HCV genomes in multiple infection models, and multiplexing of positive and negative strand vRNAs and host antiviral RNAs. We capture the varying kinetics with which antiviral drugs with different mechanisms of action clear HCV infection, finding the NS5A inhibitor daclatasvir to induce a rapid decline in negative-strand viral RNAs. We also find that the induction of host antiviral genes upon interferon treatment is positively correlated with viral load in single cells. This study adds smFISH to the toolbox available for analyzing the treatment of RNA virus infections.


Assuntos
Genoma Viral , Hepacivirus/genética , Hepatite C/virologia , Imagem Molecular , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Hepacivirus/efeitos dos fármacos , Hepatite C/diagnóstico por imagem , Hepatite C/tratamento farmacológico , Interações Hospedeiro-Patógeno/genética , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , RNA Viral , Análise de Célula Única/métodos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
9.
Cell ; 163(3): 746-58, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26496612

RESUMO

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.


Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/genética , Fármacos Anti-HIV/química , Sequência de Carboidratos , Engenharia Genética , Mitógenos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Musa/química
10.
Mol Cell ; 58(3): 541-548, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25891073

RESUMO

The vertebrate antiviral innate immune system is often considered to consist of two distinct groups of proteins: pattern recognition receptors (PRRs) that detect viral infection and induce the interferon (IFN) signaling, and effectors that directly act against viral replication. Accordingly, previous studies on PRRs, such as RIG-I and MDA5, have primarily focused on their functions in viral double-stranded RNA (dsRNA) detection and consequent antiviral signaling. We report here that both RIG-I and MDA5 efficiently displace viral proteins pre-bound to dsRNA in a manner dependent on their ATP hydrolysis, and that this activity assists a dsRNA-dependent antiviral effector protein, PKR, and allows RIG-I to promote MDA5 signaling. Furthermore, truncated RIG-I/MDA5 lacking the signaling domain, and hence the IFN stimulatory activity, displaces viral proteins and suppresses replication of certain viruses in an ATP-dependent manner. Thus, this study reveals novel "effector-like" functions of RIG-I and MDA5 that challenge the conventional view of PRRs.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Antivirais/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Fosforilação , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Receptores de Reconhecimento de Padrão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/genética , Proteínas Virais/metabolismo , Viroses/genética , Viroses/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
11.
Cell ; 160(4): 631-643, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25679759

RESUMO

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.


Assuntos
Vírus da Influenza A/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Serpina E2/metabolismo , Animais , Linhagem Celular , Humanos , Imunidade Inata , Camundongos Endogâmicos C57BL , Inibidor 1 de Ativador de Plasminogênio/genética , Sistema Respiratório/enzimologia , Sistema Respiratório/virologia , Serina Proteases/metabolismo , Serpina E2/genética
12.
Trends Immunol ; 36(3): 124-38, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25704559

RESUMO

Over half a century has passed since interferons (IFNs) were discovered and shown to inhibit virus infection in cultured cells. Since then, researchers have steadily brought to light the molecular details of IFN signaling, catalogued their pleiotropic effects on cells, and harnessed their therapeutic potential for a variety of maladies. While advances have been plentiful, several fundamental questions have yet to be answered and much complexity remains to be unraveled. We explore the current knowledge surrounding four main questions: are type I IFN subtypes differentially produced in response to distinct pathogens? How are IFN subtypes distinguished by cells? What are the mechanisms and consequences of viral antagonism? Lastly, how can the IFN response be harnessed to improve vaccine efficacy?


Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Interleucinas/imunologia , Linfócitos/imunologia , Vírus/imunologia , Imunidade Adaptativa , Evolução Biológica , Linhagem da Célula/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Evasão da Resposta Imune , Interferon Tipo I/classificação , Interferon Tipo I/genética , Interleucinas/classificação , Interleucinas/genética , Linfócitos/virologia , Filogenia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , Vírus/patogenicidade
13.
Sci Signal ; 7(327): ra50, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24866020

RESUMO

Type I interferons (IFNs), including various IFN-α isoforms and IFN-ß, are a family of homologous, multifunctional cytokines. IFNs activate different cellular responses by binding to a common receptor that consists of two subunits, IFNAR1 and IFNAR2. In addition to stimulating antiviral responses, they also inhibit cell proliferation and modulate other immune responses. We characterized various IFNs, including a mutant IFN-α2 (IFN-1ant) that bound tightly to IFNAR2 but had markedly reduced binding to IFNAR1. Whereas IFN-1ant stimulated antiviral activity in a range of cell lines, it failed to elicit immunomodulatory and antiproliferative activities. The antiviral activities of the various IFNs tested depended on a set of IFN-sensitive genes (the "robust" genes) that were controlled by canonical IFN response elements and responded at low concentrations of IFNs. Conversely, these elements were not found in the promoters of genes required for the antiproliferative responses of IFNs (the "tunable" genes). The extent of expression of tunable genes was cell type-specific and correlated with the magnitude of the antiproliferative effects of the various IFNs. Although IFN-1ant induced the expression of robust genes similarly in five different cell lines, its antiviral activity was virus- and cell type-specific. Our findings suggest that IFN-1ant may be a therapeutic candidate for the treatment of specific viral infections without inducing the immunomodulatory and antiproliferative functions of wild-type IFN.


Assuntos
Regulação da Expressão Gênica/imunologia , Interferon Tipo I/imunologia , Receptor de Interferon alfa e beta/metabolismo , Viroses/imunologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Análise por Conglomerados , Citometria de Fluxo , Humanos , Interferon Tipo I/metabolismo , Análise de Componente Principal , RNA Interferente Pequeno/genética
14.
Proc Natl Acad Sci U S A ; 108(37): 15366-71, 2011 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-21896751

RESUMO

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.


Assuntos
Antivirais/farmacologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Hidrodinâmica , Camundongos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Orthomyxoviridae/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Bibliotecas de Moléculas Pequenas/uso terapêutico , Soluções
15.
Proc Natl Acad Sci U S A ; 108(14): 5777-82, 2011 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-21436031

RESUMO

Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo pyrimidine biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo pyrimidine biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting pyrimidine pools, which are crucial for efficient virus replication.


Assuntos
Antivirais/farmacologia , Indóis/farmacologia , Oxidiazóis/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pirimidinas/biossíntese , Replicação Viral/efeitos dos fármacos , Vírus/efeitos dos fármacos , Animais , Autorradiografia , Carbamatos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Primers do DNA/genética , Humanos , Lamivudina , Mamíferos , Nevirapina , Pirimidinas/metabolismo , Pirrolidinonas , Raltegravir Potássico , Especificidade da Espécie , Sulfonamidas , Fatores de Tempo , Vírus/crescimento & desenvolvimento , beta-Galactosidase/metabolismo
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