RESUMO
The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.
Assuntos
Caenorhabditis elegans/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Repressoras/metabolismo , Envelhecimento/metabolismo , Animais , Autofagossomos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Membrana/metabolismo , ProibitinasRESUMO
Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.
Assuntos
Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Animais , Autofagia , Embrião de Mamíferos/citologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Mitofagia , Espécies Reativas de Oxigênio/metabolismo , Sindbis virus/metabolismoRESUMO
Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , FosforilaçãoRESUMO
Fanconi anemia (FA) is a rare disease, in which homozygous or compound heterozygous inactivating mutations in any of 21 genes lead to genomic instability, early-onset bone marrow failure and increased cancer risk. The FA pathway is essential for DNA damage response (DDR) to DNA interstrand crosslinks. However, proteins of the FA pathway have additional cytoprotective functions that may be independent of DDR. We have shown that many FA proteins participate in the selective autophagy pathway that is required for the destruction of unwanted intracellular constituents. In this Cell Science at a Glance and the accompanying poster, we briefly review the role of the FA pathway in DDR and recent findings that link proteins of the FA pathway to selective autophagy of viruses and mitochondria. Finally, we discuss how perturbations in FA protein-mediated selective autophagy may contribute to inflammatory as well as genotoxic stress.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Animais , Autofagia/genética , Reparo do DNA/genética , Instabilidade Genômica/genética , Humanos , Mutação/fisiologia , Transdução de Sinais/fisiologiaRESUMO
PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease-associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13-mediated mitophagy may contribute to ZSS pathogenesis.
Assuntos
Autofagia , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitofagia , Peroxissomos/metabolismo , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Sindbis virus/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Zellweger/genética , Síndrome de Zellweger/metabolismoRESUMO
The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat-beclin 1-derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef-is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat-beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/uso terapêutico , Autofagia/efeitos dos fármacos , Proteínas de Membrana/química , Proteínas de Membrana/uso terapêutico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Proteína Beclina-1 , Permeabilidade da Membrana Celular , Células Cultivadas , Vírus Chikungunya/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , HIV-1/fisiologia , Células HeLa , Humanos , Macrófagos/citologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus do Nilo Ocidental/efeitos dos fármacos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Herpesvirus Humano 8/fisiologia , Lisossomos/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/fisiologia , Proteína Beclina-1 , Transformação Celular Viral , Cruzamentos Genéticos , Modelos Animais de Doenças , Endocitose/fisiologia , Predisposição Genética para Doença , Células HEK293 , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Heterozigoto , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-6/biossíntese , Interleucina-6/genética , Lisossomos/virologia , Camundongos , NF-kappa B/metabolismo , Proteínas/fisiologia , Proteólise , RNA Interferente Pequeno , Receptores de Quimiocinas/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologiaRESUMO
Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.
Assuntos
Autofagia/genética , Estudo de Associação Genômica Ampla , RNA Interferente Pequeno/genética , Animais , Proteínas do Capsídeo/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Transporte Proteico/genética , Sindbis virus/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.
Assuntos
Autofagia/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Peptídeos Penetradores de Células/farmacologia , Proteínas do Tecido Nervoso/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Isquemia Encefálica/patologia , Glicosídeos Cardíacos/farmacologia , Células HeLa , Humanos , Ratos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Autophagy is a conserved catabolic stress response pathway that is increasingly recognized as an important component of both innate and acquired immunity to pathogens. The activation of autophagy during infection not only provides cell-autonomous protection through lysosomal degradation of invading pathogens (xenophagy), but also regulates signaling by other innate immune pathways. This review will focus on recent advances in our understanding of three major areas of the interrelationship between autophagy and innate immunity, including how autophagy is triggered during infection, how invading pathogens are targeted to autophagosomes, and how the autophagy pathway participates in "tuning" the innate immune response.
Assuntos
Autofagia , Imunidade Inata , Infecções/imunologia , Animais , Humanos , Transdução de SinaisRESUMO
Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.
Assuntos
Anemia de Fanconi , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Mitofagia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products, the autophagy (ATG) proteins. However, the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood. Here we show that two related stress-responsive kinases, members of the p38 mitogen-activated protein kinase (MAPK) signaling pathway MAPKAPK2 (MK2) and MAPKAPK3 (MK3), positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein, Beclin 1, at serine 90, and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1. Moreover, MK2/MK3-dependent Beclin 1 phosphorylation (and starvation-induced autophagy) is blocked in vitro and in vivo by BCL2, a negative regulator of Beclin 1. Together, these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation, and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteína Beclina-1 , Linhagem Celular Tumoral , Meios de Cultura , Genes Supressores de Tumor , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
The mammalian intestine is colonized with a diverse community of bacteria that perform many beneficial functions but can threaten host health upon tissue invasion. Epithelial cell-intrinsic innate immune responses are essential to limit the invasion of both commensal and pathogenic bacteria and maintain beneficial host-bacterial relationships; however, little is known about the role of various cellular processes, notably autophagy, in controlling bacterial interactions with the intestinal epithelium in vivo. We demonstrate that intestinal epithelial cell autophagy protects against tissue invasion by both opportunistically invasive commensals and the invasive intestinal pathogen Salmonella Typhimurium. Autophagy is activated following bacterial invasion of epithelial cells through a process requiring epithelial cell-intrinsic signaling via the innate immune adaptor protein MyD88. Additionally, mice deficient in intestinal epithelial cell autophagy exhibit increased dissemination of invasive bacteria to extraintestinal sites. Thus, autophagy is an important epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria.
Assuntos
Autofagia , Células Epiteliais/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Mucosa Intestinal/imunologia , Salmonelose Animal/imunologia , Animais , Modelos Animais de Doenças , Enterococcus faecalis/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Imunidade Inata , Camundongos , Salmonelose Animal/microbiologia , Salmonella typhimurium/imunologiaRESUMO
We recently identified physical exercise as a newly defined inducer of autophagy in vivo. Exercise induced autophagy in multiple organs involved in metabolic regulation, such as muscle, liver, pancreas and adipose tissue. To study the physiological role of exercise-induced autophagy, we generated mice with a knock-in nonphosphorylatable mutation in BCL2 (Thr69Ala, Ser70Ala and Ser84Ala) (BCL2 AAA) that are defective in exercise- and starvation-induced autophagy but not in basal autophagy. We found that BCL2 AAA mice could not run on a treadmill as long as wild-type mice, and did not undergo exercise-mediated increases in skeletal glucose muscle uptake. Unlike wild-type mice, the BCL2 AAA mice failed to reverse high-fat diet-induced glucose intolerance after 8 weeks of exercise training, possibly due to defects in signaling pathways that regulate muscle glucose uptake and metabolism during exercise. Together, these findings suggested a hitherto unknown important role of autophagy in mediating exercise-induced metabolic benefits. In the present addendum, we show that treadmill exercise also induces autophagy in the cerebral cortex of adult mice. This observation raises the intriguing question of whether autophagy may in part mediate the beneficial effects of exercise in neurodegeneration, adult neurogenesis and improved cognitive function.
Assuntos
Autofagia , Encéfalo/patologia , Especificidade de Órgãos , Condicionamento Físico Animal , Animais , Encéfalo/metabolismo , Exercício Físico , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Resistência Física , RatosRESUMO
In recent years, the process of selective autophagy has received much attention with respect to the clearance of protein aggregates, damaged mitochondria and bacteria. However, until recently, there have been virtually no studies on the selective autophagy of viruses, although they are perhaps one of the most ubiquitous unwanted constituents in human cells. Recently, we have shown that the ability of neuronal Atg5 to protect against lethal Sindbis virus central nervous system (CNS) infection in mice is associated with impaired viral capsid clearance, increased p62 accumulation and increased neuronal cell death. In vitro, we showed that p62 interacts with the Sindbis capsid protein and targets it for degradation in autophagosomes. Herein, we review these findings and broadly speculate about potential roles of selective viral autophagy in the regulation of host immunity and viral pathogenesis.
Assuntos
Autofagia , Vírus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Capsídeo/metabolismo , Humanos , Imunidade/imunologia , Modelos BiológicosAssuntos
Autofagia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Inflamação/metabolismo , Autofagia/genética , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Humanos , Inflamação/genéticaRESUMO
Autophagy functions in antiviral immunity. However, the ability of endogenous autophagy genes to protect against viral disease in vertebrates remains to be causally established. Here, we report that the autophagy gene Atg5 function is critical for protection against lethal Sindbis virus (SIN) infection of the mouse central nervous system. Inactivating Atg5 in SIN-infected neurons results in delayed clearance of viral proteins, increased accumulation of the cellular p62 adaptor protein, and increased cell death in neurons, but the levels of viral replication remain unaltered. In vitro, p62 interacts with SIN capsid protein, and genetic knockdown of p62 blocks the targeting of viral capsid to autophagosomes. Moreover, p62 or autophagy gene knockdown increases viral capsid accumulation and accelerates virus-induced cell death without affecting virus replication. These results suggest a function for autophagy in mammalian antiviral defense: a cell-autonomous mechanism in which p62 adaptor-mediated autophagic viral protein clearance promotes cell survival.
Assuntos
Infecções por Alphavirus/imunologia , Autofagia , Sistema Nervoso Central/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Sindbis virus/imunologia , Animais , Apoptose , Proteína 5 Relacionada à Autofagia , Linhagem Celular , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Neurônios/patologia , Neurônios/virologia , Análise de SobrevidaRESUMO
Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.
Assuntos
Hepacivirus/imunologia , Hepacivirus/fisiologia , RNA Helicases/metabolismo , RNA Viral/biossíntese , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Hepacivirus/genética , Hepacivirus/patogenicidade , Humanos , Fator Regulador 3 de Interferon , Modelos Biológicos , Mutagênese Sítio-Dirigida , RNA Helicases/química , RNA Helicases/genética , RNA Interferente Pequeno/genética , Receptores Imunológicos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Hepatitis C virus (HCV) is a major human pathogen that infects 170 million people. A hallmark of HCV is its ability to establish persistent infections reflecting the evasion of host immunity and interference with alpha/beta-IFN innate immune defenses. We demonstrate that disruption of retinoic acid-inducible gene I (RIG-I) signaling by the viral NS3/4A protease contributes to the ability of HCV to control innate antiviral defenses. RIG-I was essential for virus or HCV RNA-induced signaling to the IFN-beta promoter in human hepatoma cells. This signaling was disrupted by the protease activity of NS3/4A, which ablates RIG-I signaling of downstream IFN regulatory factor 3 and NF-kappaB activation, attenuating expression of host antiviral defense genes and interrupting an IFN amplification loop that otherwise suppresses HCV replication. Treatment of cells with an active site inhibitor of the NS3/4A protease relieved this suppression and restored intracellular antiviral defenses. Thus, NS3/4A control of RIG-I supports HCV persistence by preventing IFN regulatory factor 3 and NF-kappaB activation. Our results demonstrate that these processes are amenable to restoration through pharmacologic inhibition of viral protease function.
Assuntos
Hepacivirus/imunologia , RNA Helicases/fisiologia , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/fisiologia , Linhagem Celular Tumoral , Proteína DEAD-box 58 , RNA Helicases DEAD-box , Hepacivirus/fisiologia , Humanos , NF-kappa B/metabolismo , Receptores Imunológicos , Replicação ViralRESUMO
Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.