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At least 667 records · Page 37Linked to original sources
PubMed2001

Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system.

RNA interference (RNAi) is a gene silencing mechanism that can be triggered by introducing double-stranded RNA (dsRNA) into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. RNAi can be used for determining gene function and creating functional "knockout" organisms. Here we show for the first time that RNAi can be induced in adult fruit flies by injecting dsRNA into the abdomen of anesthetized Drosophila, and that this method can also target genes expressed in the central nervous system (CNS). Two genes were targeted to investigate the effects of dsRNA injection on their mRNA content; lacZ transgene (expressed either in the gut or in the CNS), and GM06434, the Drosophila homologue of the C. elegans gene nrf (nose resistant to fluoxetine). Both the transgene and the endogenous gene were successfully silenced in adult Drosophila by intra-abdominal injection of their respective dsRNA. We propose that our method of RNAi in adult flies can be used to characterize gene functioning in the CNS without the typical interference in development found in most gene mutation studies.

Abdomen↗
PubMed1976

The reovirus replicative cycle.

Soon after entry into their host cells ssRNA viruses form doule-stranded replicative intermediates. Most RNA viruses exist only transiently in this double-stranded form. Double-strand formation occurs irrespective of whether the genome is of the same or complementary polarity to viral mRNA. In contrast, reoviruses contain dsRNAs from the outset. Single, and not double, strands are the intracellular intermediates through which genetic information is transferred from parental to progeny reoviruses. Double strands are the repositories in which the information is stored. We emphasize this distinction because it permits dsRNA-containing viruses to replicate conservatively, a mode of replication that is not shared by any other viruses. One important consequence of the conservative mode of replication is that cellular enzymes never gain access to the reovirus genome but only to its ssRNA precursors.

Binding Sites↗
PubMed2004

Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity.

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.

Animals↗
PubMed2004

[Toll-like receptors that sense viral infection].

Anti-viral host defense harbors a variety of strategies to coup with viral infection. Recent findings suggested that Toll-like receptors (TLRs) and their signaling pathways involve type I IFN induction in response to virus-specific molecular patterns. TLR 3 and TLR 4 in myeloid dendritic cells (mDCs) recognize viral dsRNA and putative viral products, respectively, to induce IFN-beta via IRF-3 activation. On the other hand, TLR 7 and TLR 9 in plasmacytoid DCs (pDCs) induce IFN-alpha in response to their ligands, U/G-rich ssRNA and non-methylated CpG DNA. We identified TICAM-1 which is recruited to the cytoplasmic domain (designated TIR) of TLR 3 and allows to select the pathway to activation of IRF-3. We also identified TICAM-2 which binds TLR 4 and together with TICAM-1 activates IRF-3. TICAM-1 knockdown by RNAi supported the key role of TICAM-1 in IFN-beta induction. Hence, the IFN-beta induction in mDCs appears in part due to the function of TICAM-1. Viruses are known to activate kinases that directly activate IRF-3 inside the cells, and this pathway may merge with the TLR 3-TICAM-1 pathway. Here we review the relationship between the TLR 3-TICAM-1 pathway and viral infection.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

IFN-alpha sensitizes human umbilical vein endothelial cells to apoptosis induced by double-stranded RNA.

The ability of endothelial cells to mount an efficient antiviral response is important in restricting viral dissemination and eliminating viral infection from the endothelium and surrounding tissues. We demonstrate that dsRNA, a molecular signature of viral infection, induced apoptosis in HUVEC, and priming with IFN-alpha shortened the time between when dsRNA was encountered and when apoptosis was initiated. IFN-alpha priming induced higher levels of mRNA for dsRNA-activated protein kinase, 2'5'-oligoadenylate synthetase, and Toll-like receptor 3, transcripts that encode dsRNA-responsive proteins. dsRNA induced activation of dsRNA-activated protein kinase and nuclear translocation of transcription factors RelA and IFN regulatory factor-3 in IFN-alpha-primed HUVECs before the activation of intrinsic and extrinsic apoptotic pathways. These changes did not occur in the absence of dsRNA, and apoptosis resulting from incubation with dsRNA occurred much later when cells were not primed with IFN-alpha. The entire population of IFN-alpha-primed HUVECs underwent nuclear translocation of RelA and IFN regulatory factor-3 in response to dsRNA, whereas less than one-half of the population responded with apoptosis. When IFN-alpha-primed HUVECs were coincubated with dsRNA and proteasome inhibitors, all HUVECs were rendered susceptible to dsRNA-induced apoptosis. These studies provide evidence that many endothelial cells that are alerted to the risk of infection by IFN-alpha would undergo apoptosis sooner in response to dsRNA than non-IFN-alpha-primed cells, and this would enhance the likelihood of eliminating infected cells prior to the production of progeny virions.

Active Transport, Cell Nucleus↗
PubMed2006

Inverse regulation of inducible nitric oxide synthase (iNOS) and arginase I by the protein tyrosine phosphatase SHP-1 in CNS glia.

We have previously shown that the SH2 domain-containing protein tyrosine phosphatase SHP-1 plays a critical role in controlling virus infection in CNS glia in vivo and in vitro. The present study addressed whether increased virus replication in SHP-1-deficient glia in vitro may be a result of altered expression of inducible nitric oxide synthase (iNOS/NOS2). First, we observed a profound reduction in iNOS protein expression and production of nitric oxide (NO) in response to the viral mimic double-stranded RNA (dsRNA), despite the induction of high levels of iNOS mRNA, in SHP-1-deficient motheaten mouse compared to wild type littermate mouse glia. Because both iNOS expression and NO production are suppressed by multiple pathways involving arginase I activity, it was important that we observed abnormally high constitutive expression of arginase I in cultured glia of SHP-1-deficient compared to wild type mice. Further, both constitutive and IL-4/IL-10-induced expression of arginase I correlated with elevated STAT6 nuclear binding activity, decreased NO production, and increased virus replication in motheaten compared to wild type astrocytes. These findings provide the first evidence of an inverse relationship between NO and arginase I activity regulated by SHP-1 in CNS glia that is relevant to modulation of innate anti-viral responses. Thus, we propose that SHP-1 is a critical regulator of innate immunity to virus infections in CNS cells.

Animals↗
PubMed2006

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging.

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.

Bacteriophage phi 6↗
PubMed1984

Expression of the anti-retrovirus action of interferon in human cells which exhibit a reduced number of receptors for interferon.

The anti-viral and anti-cell fusion actions of human gamma interferon (IFN) were examined on human rhabdomyosarcoma cells and compared with the actions of IFN-alpha. Treatment of A204 and RD114-C1 cells with IFN-gamma resulted in significant inhibition of retrovirus production and cell fusions which were induced by Sendai virus, but IFN-gamma did not induce 2'-5' oligoadenylate (2-5A) synthetase or dsRNA-dependent protein kinase, and failed to inhibit EMC virus replication in RD114-C1 cells as previously observed on IFN-alpha treatment (Tomita, Y. et al. (1982) Virology 120, 258-263). Although IFN-gamma induced 56K protein more strongly than IFN-alpha in human transformed HEp-2, HeLa, RSa, IFr, and A204 cells, no significant induction of this protein was observed in RD114-C1 cells after IFN-alpha or IFN-gamma treatment. Specific bindings of 125I-labeled human IFN-alpha A to HeLa, A204 and RD114-C1 cell surfaces showed that the numbers of the binding sites on RD114-C1 cells were reduced to less than 22% of those on A204 cells. These results suggest that RD114-C1 cells exhibit a reduced number of receptors for IFN on the cell surface and that the receptors are functional for the expression of the anti-retrovirus and anti-cell fusion actions of IFN, but are not enough in number for expression of the anti-EMC virus action of IFN.

Antiviral Agents↗
PubMed2001

PACT and PKR: turning on NF-kappa B in the absence of virus.

Double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) has been generally thought to be solely regulated by dsRNA, an intermediate in the replication of many viruses. However, the notion that PKR acts solely as a sensor for viral infection has been challenged by recent findings that alteration of PKR activity has effects on cellular growth and by the discovery of a virus-independent activator of PKR, a cellular protein called PACT (PKR-activating protein). The activation of the transcription factor nuclear factor kappa B (NF-kappaB) by PKR has been shown to account for the host antiviral response. We summarize the most recent findings on the molecular mechanisms leading to the activation of NF-kappaB by PKR and discuss three major unanswered questions. First, is PACT an alternative to dsRNA as a direct activator of the PKR-NF-kappaB pathway? Second, how is PACT itself activated and targeted to PKR? And third, what are the biological functions of PKR in the absence of viral infection?

Animals↗
PubMed2005

Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif.

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Regulation of CXCL-8 (interleukin-8) induction by double-stranded RNA signaling pathways during hepatitis C virus infection.

Hepatitis C virus (HCV) infection induces the alpha-chemokine interleukin-8 (CXCL-8), which is regulated at the levels of transcription and mRNA stability. In the current study, CXCL-8 regulation by double-stranded (ds)RNA pathways was analyzed in the context of HCV infection. A constitutively active mutant of the retinoic acid-inducible gene I (RIG-I), RIG-N, activated CXCL-8 transcription. Promoter mutagenesis experiments indicated that NF-kappaB and interferon (IFN)-stimulated response element (ISRE) binding sites were required for the RIG-N induction of CXCL-8 transcription. IFN-beta promoter stimulator 1 (IPS-1) expression also activated CXCL-8 transcription, and mutations of the ISRE and NF-kappaB binding sites reduced and abrogated CXCL-8 transcription, respectively. In the presence of wild-type RIG-I, transfection of JFH-1 RNA or JFH-1 virus infection of Huh7.5.1 cells activated the CXCL-8 promoter. Expression of IFN regulatory factor 3 (IRF-3) stimulated transcription from both full-length and ISRE-driven CXCL-8 promoters. Chromatin immunoprecipitation assays demonstrated that IRF-3 and NF-kappaB bound directly to the CXCL-8 promoter in response to virus infection and dsRNA transfection. RIG-N stabilized CXCL-8 mRNA via the AU-rich element in the 3' untranslated region of CXCL-8 mRNA, leading to an increase in its half-life following tumor necrosis factor alpha induction. The data indicate that HCV infection triggers dsRNA signaling pathways that induce CXCL-8 via transcriptional activation and mRNA stabilization and define a regulatory link between innate antiviral and inflammatory cellular responses to virus infection.

Cell Line↗
PubMed2004

Single processing center models for human Dicer and bacterial RNase III.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.

Amino Acid Sequence↗
PubMed2002

Virion disassembly is required for apoptosis induced by reovirus.

Reovirus infection leads to apoptosis in cultured cells and in vivo. Binding of viral attachment protein final sigma 1 to both sialic acid and junction adhesion molecule is required for induction of apoptosis. However, it is not known whether viral engagement of receptors is sufficient to elicit this cellular response. To determine whether steps in reovirus replication subsequent to viral attachment are required for reovirus-induced apoptosis, we used inhibitors of viral disassembly and RNA synthesis, viral disassembly intermediates, temperature-sensitive (ts) reovirus mutants, and reovirus particles deficient in genomic double-stranded RNA (dsRNA). We found that reovirus-induced apoptosis is abolished in the presence of the viral disassembly inhibitors ammonium chloride and E64. Infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly that can be generated in vitro by protease treatment, are capable of inducing apoptosis in the presence or absence of these inhibitors. Treatment of cells with the viral RNA synthesis inhibitor ribavirin does not diminish the capacity of reovirus to induce apoptosis, and reovirus ts mutants arrested at defined steps in viral replication produce apoptosis with efficiency similar to that of wild-type virus. Furthermore, reovirus particles lacking dsRNA are capable of inducing apoptosis. Finally, we found that viral attachment and disassembly must occur within the same cellular compartment for reovirus to elicit an apoptotic response. These results demonstrate that disassembly of reovirus virions to form ISVPs, but not viral transcription or subsequent steps in viral replication, is required for reovirus to induce apoptosis.

Ammonium Chloride↗
PubMed2001

Specific cleavage of hyper-edited dsRNAs.

Extended double-stranded DNA (dsRNA) duplexes can be hyper-edited by adenosine deaminases that act on RNA (ADARs). Long uninterrupted dsRNA is relatively uncommon in cells, and is frequently associated with infection by DNA or RNA viruses. Moreover, extensive adenosine to inosine editing has been reported for various viruses. A number of cellular antiviral defence strategies are stimulated by dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. We describe here a cytoplasmic endonuclease activity that specifically cleaves hyper-edited dsRNA. Cleavage occurs at specific sites consisting of alternating IU and UI base pairs. In contrast, unmodified dsRNA and even deaminated dsRNAs that contain four consecutive IU base pairs are not cleaved. Moreover, dsRNAs in which alternating IU and UI base pairs are replaced by isomorphic GU and UG base pairs are not cleaved. Thus, the cleavage of deaminated dsRNA appears to require an RNA structure that is unique to hyper-edited RNA, providing a molecular target for the disposal of hyper-edited viral RNA.

Adenosine Deaminase↗
PubMed1995

Complete nucleotide sequences of 5'- and 3'-noncoding regions of both genome segments of different strains of infectious bursal disease virus.

The terminal sequences of both genomic dsRNA segments of different strains of infectious bursal disease virus were determined. Analysis of the linear sequences revealed in the 5'-noncoding region of both segments the presence of a 32-nucleotide sequence with motifs conserved between segments and serotypes. The 3'-noncoding regions showed a high degree of conservation within segment A or B, but differed between segments. Different inverted terminal repeats were detected in both segments. A sequence of 13 nucleotides found in the 5'-noncoding regions of both segments could function as a binding site for chicken 18 S rRNA. Secondary structure prediction of the different noncoding regions showed a high degree of similarity. Surprisingly, the predicted secondary structure of the 5'-noncoding region of segment A differed between serotypes. A possible influence of these different RNA structures on viral replication is discussed.

Animals↗
PubMed1989

Studies on the mechanism of the antiviral activity of ribavirin against reovirus.

We have examined the mechanism by which ribavirin inhibits the multiplication of reovirus. At a concentration of 12.5 microM (3 micrograms/ml) ribavirin inhibits viral multiplication, ssRNA formation, dsRNA formation, and protein synthesis by about 90%; when much higher concentrations are used for brief periods of time, the primary target of ribavirin is seen to be viral ssRNA synthesis. When the effect of ribavirin triphosphate (RTP) was tested on the in vitro transcription by cores of the dsRNA genome segments into plus-stranded RNA, elongation, that is, the formation of intact mRNA molecules, was found to be inhibited to the greatest extent; initiation was at least 2.5 times less sensitive, and cap formation and methylation were almost unaffected. The inhibition of elongation and initiation was not competitive with respect to any of the four nucleoside triphosphates. Remarkably, the transcription of plus strands into minus strands by immature reovirus particles (the replicase reaction) was insensitive to RTP. A model is proposed that envisages RTP binding to a site close to the catalytic site of the transcriptase. This binding is postulated to inhibit the helicase function of the transcriptase and lower its affinity for template RNA so that the likelihood of premature termination is greatly increased. The helicase activity is not, of course, necessary for the transcription of plus strands into minus strands, which would account for the differential sensitivity of the transcriptase and the replicase to RTP.

Animals↗
PubMed2005

Retention and repression: fates of hyperedited RNAs in the nucleus.

Double-stranded RNA (dsRNA) is often formed in the nuclei of mammalian cells, but in this compartment it does not induce the effects characteristic of cytoplasmic dsRNA. Rather, recent work has suggested that nuclear dsRNA is a target for the ADAR class of enzymes, which deaminate adenosines to inosines. Further, there are a number of distinct fates of such edited RNA, including nuclear retention and perhaps also gene silencing.

Active Transport, Cell Nucleus↗
PubMed2001

Double-stranded rna dependence of nitric oxide synthase 2 expression in human bronchial epithelial cell lines BET-1A and BEAS-2B.

The human airway epithelium expresses abundant nitric oxide synthase 2 (NOS2) in vivo. Although NOS2 is easily induced by cytokines in primary cultured human airway epithelial cells and lung adenocarcinoma cell line A549, the human bronchial epithelial cell lines BEAS-2B and BET-1A do not express NOS2 in response to cytokines. Mechanisms regulating NOS2 expression in human respiratory epithelial cells are complex, but we have recently shown that NOS2 expression in primary human airway epithelial cells occurs in response to double-stranded RNA (dsRNA) through activation of signaling proteins including nuclear factor (NF)-kappaB and interferon (IFN) regulatory factor (IRF)-1. In this context, we hypothesized that BEAS-2B and BET-1A cells may express NOS2 in response to dsRNA. Here, we show that although cytokines (IFN-gamma, tumor necrosis factor-alpha and interleukin-1beta) do not induce NOS2 expression in BEAS-2B or BET-1A cells, addition of dsRNA to this cytokine mix enables BEAS-2B cells to express NOS2. IFN-gamma and dsRNA induction of NOS2 in BET-1A cells occurs in a serum concentration-dependent manner, with a minimum of 3 d of serum treatment necessary for BET-1A cells to acquire the potential to induce NOS2. Importantly, dsRNA strongly activates NF-kappaB and IRF-1 in BEAS-2B cells, transcription factors essential for NOS2 gene expression in other cell lines. On the basis of these results, dsRNA-activated signaling pathways are clearly important for NOS2 expression in human respiratory epithelial cells. With conditions for NOS2 expression characterized, these cell lines are a convenient in vitro system to investigate the mechanisms regulating NOS2 expression in human respiratory epithelial cells.

Bronchi↗