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1.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295917

RESUMO

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen-associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount an effective host response during viral infections.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Endorribonucleases/metabolismo , eIF-2 Quinase/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Endorribonucleases/fisiologia , Humanos , Interferon beta/genética , Interferons/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/genética , eIF-2 Quinase/fisiologia
2.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31330998

RESUMO

Apoptosis of virus-infected cells is an effective antiviral mechanism in addition to interferon induction to establish antiviral state to restrict virus spread. The interferon-inducible 2'-5' oligoadenylate synthetase/RNase L pathway results in activation of RNase L in response to double stranded RNA and cleaves diverse RNA substrates to amplify interferon induction and promote apoptosis. Here we show that RNase L induces expression of Death-associated protein kinase-Related Apoptosis-inducing protein Kinase 1 (DRAK1), a member of the death-associated protein kinase family and interferon-signaling pathway is required for induction. Overexpression of DRAK1 triggers apoptosis in the absence of RNase L activation by activating c-Jun N-terminal kinase (JNK), translocation of BCL2 Associated X (Bax) to the mitochondria accompanied by cytochrome C release and loss of mitochondrial membrane potential promoting cleavage of caspase 3 and Poly(ADP-Ribose) Polymerase 1 (PARP). Inhibitors of JNK and caspase 3 promote survival of DRAK1 overexpressing cells demonstrating an important role of JNK signaling pathway in DRAK1-mediated apoptosis. DRAK1 mutant proteins that lack kinase activity or nuclear localization fail to induce apoptosis highlighting the importance of cellular localization and kinase function in promoting cell death. Our studies identify DRAK1 as a mediator of RNase L-induced apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Expressão Ectópica do Gene , Humanos , Interferons/metabolismo , Interferons/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Transdução de Sinais
3.
J Virol ; 91(19)2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28747493

RESUMO

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.


Assuntos
Septicemia Hemorrágica Viral/patologia , Novirhabdovirus/crescimento & desenvolvimento , Novirhabdovirus/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Replicação Viral/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Cyprinidae , Doenças dos Peixes/virologia , Células HEK293 , Septicemia Hemorrágica Viral/virologia , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Fosforilação/genética , Regiões Promotoras Genéticas/genética , RNA/genética , RNA Polimerase II/antagonistas & inibidores , Vírus 40 dos Símios/genética , Transcrição Gênica/fisiologia
4.
Int J Mol Sci ; 18(3)2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28257035

RESUMO

The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin ß1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene.


Assuntos
Endorribonucleases/metabolismo , Metaloproteinases da Matriz/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Endorribonucleases/genética , Ativação Enzimática , Humanos , Masculino , Mutação , Neoplasias da Próstata/genética , Ligação Proteica , Estabilidade Proteica
5.
Int J Mol Sci ; 17(1)2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26760998

RESUMO

The interferon (IFN)-regulated endoribonuclease RNase-L is involved in multiple aspects of the antimicrobial innate immune response. It is the terminal component of an RNA cleavage pathway in which dsRNA induces the production of RNase-L-activating 2-5A by the 2'-5'-oligoadenylate synthetase. The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. This leads to IFNß expression and IL-1ß activation respectively, in addition to broader effects on immune cell function. RNase-L is also one of a growing number of innate immune components that interact with the cell cytoskeleton. It can bind to several cytoskeletal proteins, including filamin A, an actin-binding protein that collaborates with RNase-L to maintain the cellular barrier to viral entry. This antiviral activity is independent of catalytic function, a unique mechanism for RNase-L. We also describe here the interaction of RNase-L with the E3 ubiquitin ligase and scaffolding protein, ligand of nump protein X (LNX), a regulator of tight junction proteins. In order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed.


Assuntos
Infecções Bacterianas/imunologia , Citoesqueleto/imunologia , Citoesqueleto/microbiologia , Endorribonucleases/imunologia , Imunidade Inata , Viroses/imunologia , Animais , Bactérias/imunologia , Infecções Bacterianas/enzimologia , Humanos , Viroses/enzimologia , Vírus/imunologia
6.
Int J Mol Sci ; 16(8): 17611-36, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26263979

RESUMO

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Autofagia/genética , Endorribonucleases/genética , Proteínas de Membrana/metabolismo , Neoplasias/genética , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Endorribonucleases/metabolismo , Humanos , Infecções/genética , Infecções/patologia , Infecções/virologia , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Neoplasias/patologia , Neoplasias/virologia , RNA de Cadeia Dupla/genética
7.
J Biol Chem ; 287(52): 43651-64, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23109342

RESUMO

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.


Assuntos
Autofagia , Endorribonucleases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , eIF-2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/metabolismo , Infecções por Cardiovirus/patologia , Linhagem Celular Tumoral , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Vírus da Encefalomiocardite/fisiologia , Endorribonucleases/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Infecções por Respirovirus/genética , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/patologia , Vírus Sendai/fisiologia , Proteína Sequestossoma-1 , Replicação Viral/fisiologia , eIF-2 Quinase/genética
8.
Nature ; 448(7155): 816-9, 2007 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-17653195

RESUMO

Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode alpha- and beta-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, interferon-beta promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-kappaB to the IFN-beta gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2',5'-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-beta expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3' of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-beta expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-beta during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-beta in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential role in the innate antiviral immune response that relieves the requirement for direct sensing of non-self RNA.


Assuntos
Infecções por Cardiovirus/imunologia , Vírus da Encefalomiocardite/imunologia , Endorribonucleases/metabolismo , Imunidade Inata/imunologia , RNA/imunologia , RNA/metabolismo , Nucleotídeos de Adenina/imunologia , Animais , Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/fisiologia , Endorribonucleases/deficiência , Endorribonucleases/genética , Fibroblastos , Interferon beta/biossíntese , Interferon beta/genética , Interferon beta/imunologia , Camundongos , Oligorribonucleotídeos/imunologia , Regiões Promotoras Genéticas/genética , RNA/química , RNA de Cadeia Dupla/imunologia , Transdução de Sinais , Ativação Transcricional/genética
9.
Viruses ; 15(2)2023 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-36851680

RESUMO

Virus infection activates integrated stress response (ISR) and stress granule (SG) formation and viruses counteract by interfering with SG assembly, suggesting an important role in antiviral defense. The infection of fish cells by Viral Hemorrhagic Septicemia Virus (VHSV), activates the innate immune recognition pathway and the production of type I interferon (IFN). However, the mechanisms by which VHSV interacts with ISR pathway regulating SG formation is poorly understood. Here, we demonstrate that fish cells respond to heat shock, oxidative stress and VHSV infection by forming SG that localized key SG marker, Ras GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1). We show that PKR-like endoplasmic reticulum kinase (PERK), but not (dsRNA)-dependent protein kinase (PKR), is required for VHSV-induced SG formation. Furthermore, in VHSV Ia infected cells, PERK activity is required for IFN production, antiviral signaling and viral replication. SG formation required active virus replication as individual VHSV Ia proteins or inactive virus did not induce SG. Cells lacking G3BP1 produced increased IFN, antiviral genes and viral mRNA, however viral protein synthesis and viral titers were reduced. We show a critical role of the activation of ISR pathway and SG formation highlighting a novel role of G3BP1 in regulating VHSV protein translation and replication.


Assuntos
DNA Helicases , Novirhabdovirus , Animais , Antivirais , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Replicação Viral
10.
PNAS Nexus ; 2(10): pgad299, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37822767

RESUMO

The underlying biological mechanisms that contribute to the heterogeneity of major depressive disorder (MDD) presentation remain poorly understood, highlighting the need for a conceptual framework that can explain this variability and bridge the gap between animal models and clinical endpoints. Here, we hypothesize that comparative analysis of molecular data from different experimental systems of chronic stress, and MDD has the potential to provide insight into these mechanisms and address this gap. Thus, we compared transcriptomic profiles of brain tissue from postmortem MDD subjects and from mice exposed to chronic variable stress (CVS) to identify orthologous genes. Ribosomal protein genes (RPGs) were down-regulated, and associated ribosomal protein (RP) pseudogenes were up-regulated in both conditions. A seeded gene co-expression analysis using altered RPGs common between the MDD and CVS groups revealed that down-regulated RPGs homeostatically regulated the synaptic changes in both groups through a RP-pseudogene-driven mechanism. In vitro analysis demonstrated that the RPG dysregulation was a glucocorticoid-driven endocrine response to stress. In silico analysis further demonstrated that the dysregulation was reversed during remission from MDD and selectively responded to ketamine but not to imipramine. This study provides the first evidence that ribosomal dysregulation during stress is a conserved phenotype in human MDD and chronic stress-exposed mouse. Our results establish a foundation for the hypothesis that stress-induced alterations in RPGs and, consequently, ribosomes contribute to the synaptic dysregulation underlying MDD and chronic stress-related mood disorders. We discuss the role of ribosomal heterogeneity in the variable presentations of depression and other mood disorders.

11.
RNA ; 16(11): 2108-19, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20833746

RESUMO

Triggering and propagating an intracellular innate immune response is essential for control of viral infections. RNase L is a host endoribonuclease and a pivotal component of innate immunity that cleaves viral and cellular RNA within single-stranded loops releasing small structured RNAs with 5'-hydroxyl (5'-OH) and 3'-monophosphoryl (3'-p) groups. In 2007, we reported that RNase L cleaves self RNA to produce small RNAs that function as pathogen-associated molecular patterns (PAMPs). However, the precise sequence and structure of PAMP RNAs produced by RNase L is unknown. Here we used hepatitis C virus RNA as substrate to characterize RNase L mediated cleavage products [named suppressor of virus RNA (svRNA)] for their ability to activate RIG-I like receptors (RLR). The NS5B region of HCV RNA was cleaved by RNase L to release an svRNA that bound to RIG-I, displacing its repressor domain and stimulating its ATPase activity while signaling to the IFN-ß gene in intact cells. All three of these RIG-I functions were dependent on the presence in svRNA of the 3'-p. Furthermore, svRNA suppressed HCV replication in vitro through a mechanism involving IFN production and triggered a RIG-I-dependent hepatic innate immune response in mice. RNase L and OAS (required for its activation) were both expressed in hepatocytes from HCV-infected patients, raising the possibility that the OAS/RNase L pathway might suppress HCV replication in vivo. It is proposed that RNase L mediated cleavage of HCV RNA generates svRNA that activates RIG-I, thus propagating innate immune signaling to the IFN-ß gene.


Assuntos
Endorribonucleases/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Hepacivirus/imunologia , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , RNA Viral/imunologia , Receptores de Superfície Celular , Receptores Imunológicos , Especificidade por Substrato , Replicação Viral
12.
Viruses ; 13(2)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670646

RESUMO

Host response to a viral infection includes the production of type I interferon (IFN) and the induction of interferon-stimulated genes that have broad antiviral effects. One of the key antiviral effectors is the IFN-inducible oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathway, which is activated by double-stranded RNA to synthesize unique oligoadenylates, 2-5A, to activate RNase L. RNase L exerts an antiviral effect by cleaving diverse RNA substrates, limiting viral replication; many viruses have evolved mechanisms to counteract the OAS/RNase L pathway. Here, we show that the ATP-binding cassette E1 (ABCE1) transporter, identified as an inhibitor of RNase L, regulates RNase L activity and RNase L-induced autophagy during viral infections. ABCE1 knockdown cells show increased RNase L activity when activated by 2-5A. Compared to parental cells, the autophagy-inducing activity of RNase L in ABCE1-depleted cells is enhanced with early onset. RNase L activation in ABCE1-depleted cells inhibits cellular proliferation and sensitizes cells to apoptosis. Increased activity of caspase-3 causes premature cleavage of autophagy protein, Beclin-1, promoting a switch from autophagy to apoptosis. ABCE1 regulates autophagy during EMCV infection, and enhanced autophagy in ABCE1 knockdown cells promotes EMCV replication. We identify ABCE1 as a host protein that inhibits the OAS/RNase L pathway by regulating RNase L activity, potentially affecting antiviral effects.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Autofagia , Endorribonucleases/metabolismo , Viroses/metabolismo , Fenômenos Fisiológicos Virais , Transportadores de Cassetes de Ligação de ATP/genética , Apoptose , Endorribonucleases/genética , Humanos , Viroses/enzimologia , Viroses/fisiopatologia , Viroses/virologia , Vírus/genética
13.
Viruses ; 12(5)2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32365817

RESUMO

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Novirhabdovirus/genética , Biossíntese de Proteínas , Infecções por Rhabdoviridae/veterinária , Proteínas Virais/genética , eIF-2 Quinase/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/genética , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Peixes , Interações Hospedeiro-Patógeno , Interferons/genética , Interferons/metabolismo , Novirhabdovirus/isolamento & purificação , Novirhabdovirus/metabolismo , Fosforilação , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Proteínas Virais/metabolismo , eIF-2 Quinase/genética
14.
J Cell Biol ; 164(4): 547-56, 2004 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-14970192

RESUMO

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Esteróis/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Células CHO , Membrana Celular/metabolismo , Cricetinae , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteína C1 de Niemann-Pick , Doenças de Niemann-Pick/genética , Doenças de Niemann-Pick/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Proteínas de Transporte Vesicular
15.
PLoS Pathog ; 2(3): e25, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16609730

RESUMO

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals.


Assuntos
Adenocarcinoma/genética , Endorribonucleases/genética , Gammaretrovirus/isolamento & purificação , Predisposição Genética para Doença , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/virologia , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , DNA Viral/análise , Endorribonucleases/metabolismo , Gammaretrovirus/genética , Genoma Viral , Homozigoto , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/virologia
16.
Nucleic Acids Res ; 34(22): 6684-95, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17145707

RESUMO

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2',5'-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Neoplasias da Próstata/enzimologia , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/genética , Nucleotídeos de Adenina/química , Linhagem Celular Tumoral , Clonagem Molecular , Retrovirus Endógenos/genética , Ativação Enzimática , Produtos do Gene env/genética , Humanos , Masculino , Oligorribonucleotídeos/química , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo
17.
J Indian Soc Periodontol ; 22(5): 438-441, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30210194

RESUMO

CONTEXT: The role of synbiotic in the management of aggressive periodontitis (AP) is not well established. AIM: We designed this study to evaluate the efficacy and safety of synbiotic as an add-on therapy in the management of AP. SETTINGS AND DESIGN: This was a randomized, double-blind, placebo-controlled study. MATERIALS AND METHODS: Sixty patients with AP were randomized to two groups of 30 each. Study group received standard treatment, i.e., scaling and root planing along with doxycycline 100 mg twice daily for the 1st day (loading dose) followed by 100 mg once daily for 1 week and synbiotic lozenge twice daily for 8 weeks, whereas control group received standard treatment for 1 week and placebo lozenge for 8 weeks. Clinical parameters were recorded at baseline, 4th, 8th, and 12th weeks. STATISTICAL ANALYSIS USED: Pearson's Chi-square test and one-way analysis of variance were used to analyze distribution of sex and age, respectively. Differences within the groups were analyzed by Student's paired t-test. Pearson's Chi-square test was applied to measure gingival bleeding index. RESULTS: On comparison at 12 weeks, there was a statistically significant reduction (P < 0.01) in probing depth, clinical attachment loss, oral hygiene index, and bleeding on probing in study group. No Adverse drug reactions were noted. CONCLUSION: Co-administration of synbiotic lozenge along with standard therapy is highly efficacious in improving periodontal health in patients with AP.

18.
Cancer Res ; 64(24): 9144-51, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15604285

RESUMO

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2',5'-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH(2)-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH(2)-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/análogos & derivados , Endorribonucleases/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Inibidores da Topoisomerase I , Nucleotídeos de Adenina/administração & dosagem , Nucleotídeos de Adenina/farmacologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Sinergismo Farmacológico , Endorribonucleases/deficiência , Endorribonucleases/genética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Humanos , Irinotecano , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4 , Masculino , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligante Indutor de Apoptose Relacionado a TNF , Topotecan/administração & dosagem , Topotecan/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacologia
19.
J Interferon Cytokine Res ; 25(10): 595-603, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16241858

RESUMO

2',5'-Oligoadenylate (2-5A)-dependent RNase L is a ubiquitous endoribonuclease of higher vertebrates that functions in the interferon (IFN) antiviral response by degrading both viral and cellular single-stranded RNA (ssRNA). In addition, the RNase L gene, RNASEL, was mapped to the hereditary prostate cancer 1 (HPC1) gene. Previous analyses of human RNASEL determined its exon/intron structure but lacked a description of the promoter region. We thus mapped the RNASEL transcriptional start site using 5'-rapid amplification of cDNA ends (5'-RACE) and primer extension methods with RNA from human histiocytic lymphoma U937 cells. The promoter sequence was analyzed for potential transcription factor binding sites. Although a canonical IFN-gamma activation site (GAS) element (TTCCAAGAA) was identified (nucleotides -155 to -147), there was only slight induction of RNASEL promoter-reporter activity or of endogenous RNase L expression in response to IFN-alpha or IFN-gamma. Several sites for tissue-specific and general promoters were observed, however, which could explain the widespread expression of RNase L in mammalian cells. Accordingly, RNase L levels were determined and compared in different human and rodent cancer and normal cell types using a radiolabeled 2-5A derivative. In addition, levels of RNase L were established in various normal human tissues and cell types by immunoblotting and immunohistochemistry. Our findings are the first description of the human RNASEL promoter that allows constituitive expression in a range of normal and neoplastic cell types.


Assuntos
Endorribonucleases/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Elementos de Resposta/genética , Animais , Linhagem Celular Tumoral , Endorribonucleases/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interferon gama/metabolismo , Masculino , Camundongos , Neoplasias/genética , Neoplasias/patologia , RNA Viral/metabolismo , Viroses/metabolismo
20.
J Inflamm Res ; 7: 89-101, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24971028

RESUMO

Chronic inflammation of the prostate contributes to the increased risk of prostate cancer. Microbial pathogens in the prostate cause inflammation that leads to prostatitis and proliferative inflammatory atrophy frequently associated with the development of prostate cancer. Bacterial lipopolysaccharides and DNA mediate immune responses by engaging Toll-like receptor (TLR) 4 and 9, respectively. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) mimic bacterial DNA and signal through TLR9 to initiate innate immune responses. Here, we show that stimulation of DU145, PC3, or LnCap prostate cancer cells by the TLR9 agonists, CpG-ODN, induces mRNA expression of IL-6, IL-8, CXCL1, IP-10, CCL5, and TGFß. In addition, activity of matrix metalloproteinase (MMP)-9 and -2 and cell migration increased on CpG-ODN treatment. Induction of cytokines and chemokines was mediated by NF-κB activation and translocation to the nucleus. Treatment with epigallocatechin-3-gallate (EGCG), the major constituent of green tea, prior to CpG-ODN stimulation, inhibits cytokine and chemokine gene induction, activity of MMP-9 and -2, and cell migration. EGCG treatment sequesters the p65 subunit of transcription factor NF-κB in the cytoplasm and inhibits transcriptional activity of the NF-κB-driven promoter in response to CpG-ODN. Our results suggest that the ability of the TLR9 agonists, CpG-ODN, to induce cytokines, chemokines, and MMP activity, as well as suppression by EGCG are independent of the androgen receptor and p53 status of the cells. EGCG may provide protective effects against inflammation in the prostate and benefit prostate cancer treatment.

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