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PubMed1980

Encapsidation of yeast killer double-stranded ribonucleic acids: dependence of M on L.

Virus-like particles containing either L or M double-stranded ribonucleic acid (dsRNA) were isolated from a killer toxin-producing strains of Saccharomyces cerevisiae (K+ R+). At least 95% of M- and 87% of L-dsRNA were recovered in virus-like particle-containing fractions. The major capsid polypeptides (ScV-P1) of both L and M virus-like particles were shown to be identical, and 95% of the cellular ScV-P1 was found in the virus-like particle-containing fractions. Since L-dsRNA encodes ScV-P1, provision of this protein for encapsidation of M-dsRNA defines at least one functional relationship between these dsRNA genomes and associates the L-dsRNA with the killer character. If encapsidation of M-dsRNA is essential for its replication or expression, then L-dsRNA plays an essential role in maintenance or expression of the killer phenotype. The relationship between the L- and M-dsRNA genomes would be analogous to that between a helper and a defective virus. The presence of only minor quantities or uncomplexed dsRNA and ScV-P1 suggests that their production is stringently coupled.

Capsid↗
PubMed2006

Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein.

Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.

Animals↗
PubMed2006

Overproduction of double-stranded RNA in vesicular stomatitis virus-infected cells activates a constitutive cell-type-specific antiviral response.

In a companion paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:492-502, 2007), we provided indirect evidence that cell-type-specific growth restriction of the vesicular stomatitis virus (VSV) polR mutants may be due to enhanced production of double-stranded RNA (dsRNA). We show here that polR growth in mouse L-929 cells was rescued by vaccinia virus coinfection and that sole expression of the vaccinia virus dsRNA-binding E3L protein, via coinfection with an engineered VSV minigenome, also restored polR growth. Expression of dsRNA-binding protein NS1A or NS1B from influenza virus, but not C protein from Sendai virus, which does not bind dsRNA, likewise effected polR rescue. The N-terminal dsRNA-binding domain of NS1A, only 73 amino acids in length, but not a full-size mutant NS1A lacking dsRNA-binding activity, restored polR growth. Both key aspects of polR growth restriction, namely inhibition of genome replication and release of low-infectivity virus particles, were countered by expression of the dsRNA-binding proteins. We tested the effects of overproducing dsRNA in wild-type VSV infections by coinfecting cells with a VSV recombinant expressing the sense strand of the enhanced green fluorescent protein gene (VSV-GFP) and one expressing the antisense strand (VSV-PFG). These coinfections mimicked all aspects of polR restriction, including host range, lack of effect on transcription, reduced virus particle infectivity, and insensitivity to inhibition of host gene transcription or dsRNA-activated protein kinase activity. We conclude that, for some cell types, overproduction of dsRNA during VSV infection triggers an immediate and constitutive host cell antiviral effector response independent of interferon induction or signaling.

Animals↗
PubMed2005

Double-stranded RNA binding by human cytomegalovirus pTRS1.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes rescue replication of vaccinia virus (VV) that has a deletion of the double-stranded RNA binding protein gene E3L (VVDeltaE3L). Like E3L, these HCMV genes block the activation of key interferon-induced, double-stranded RNA (dsRNA)-activated antiviral pathways. We investigated the hypothesis that the products of these HCMV genes act by binding to dsRNA. pTRS1 expressed by cell-free translation or by infection of mammalian cells with HCMV or recombinant VV bound to dsRNA. Competition experiments revealed that pTRS1 preferentially bound to dsRNA compared to double-stranded DNA or single-stranded RNA. 5'- and 3'-end deletion analyses mapped the TRS1 dsRNA-binding domain to amino acids 74 through 248, a region of identity to pIRS1 that contains no homology to known dsRNA-binding proteins. Deletion of the majority of this region (Delta86-246) completely abrogated dsRNA binding. To determine the role of the dsRNA-binding domain in the rescue of VVDeltaE3L replication, wild-type or deletion mutants of TRS1 were transfected into HeLa cells, which were then infected with VVDeltaE3L. While full-length TRS1 rescued VVDeltaE3L replication, deletion mutants affecting a carboxy-terminal region of TRS1 that is not required for dsRNA binding failed to rescue VVDeltaE3L. Analyses of stable cell lines revealed that the carboxy-terminal domain is necessary to prevent the shutoff of protein synthesis and the phosphorylation of eIF2alpha after VVDeltaE3L infection. Thus, pTRS1 contains an unconventional dsRNA-binding domain at its amino terminus, but a second function involving the carboxy terminus is also required for countering host cell antiviral responses.

Animals↗
PubMed2006

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA.

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

Animals↗
PubMed1991

[Phosphorylation of proteins in cytoplasmic and nuclear fractions of human cells treated with double-helical RNA and human recombinant interferon type I].

The dynamics of protein kinases activity in nuclear and cytoplasmic fractions of human fibroblasts treated by preparations of natural and synthetic dsRNA (ridostin, rifastin, larifan and poly(I).poly(C), DEAE-dextran and dsRNA complexes with DEAE-dextran), as well as by preparations of recombinant alpha-2 and beta-1 interferons was obtained. The early activation of enzymes in treated cells extracts and their presence in dsRNA-activated and nonactivated forms were found. In cytoplasmic cellular fractions treated by interferons the dsRNA dependent protein kinases (nonactivated forms- were prevalent.r In contrast, in dsRNA treated cells or dsRNA complexes with DEAE-dextran treated ones the dsRNA independent protein kinases (activated forms) were found, while dsRNA dependent forms induced by interferons were found at later periods. Nuclear protein kinases are mainly dsRNA independent making possible the supposition of their intracellular activation by incoming dsRNA or interferon-induced formation of ds-structures in cellular nuclei. In phosphorylated proteins spectre the 90, 69, 45-40 and 30-35 kDa polypeptides were found. At early intervals in nuclear fractions was found a nuclease resistant and partially EDTA resistant high molecular phosphorylated complex (120 kDa). The complex is, probably, capable of dissociation to low mol mass components. DEAE-dextran induces strong activation of protein kinases in cytoplasm and nuclei and increases the content of activated forms of enzyme in larifan treated cells.

Cell Nucleus↗
PubMedDate not supplied

Differential effects of human natural interferon-alpha and mismatched double-stranded RNA against a human renal cell carcinoma xenograft.

The antitumor effects of natural human IFN-alpha and mismatched dsRNA against the human renal cell carcinoma cell line 786-0 were studied both in a clonogenic soft agar assay and in the nude mouse. The 786-0 cells were sensitive in vitro to the antiproliferative effects of IFN-alpha in a dose-response manner, up to 3000 IRU/ml. These cells were also sensitive, in a dose-dependent manner, to mismatched dsRNA in the clonogenic assay. Mismatched dsRNA was effective in inhibiting tumor growth (p less than 0.001) in nude mouse xenografts, with regression of the tumor mass seen in all animals. A significant increase in survival (p less than 0.001) was seen in the mismatched dsRNA treated group. In contrast, IFN-alpha did not inhibit tumor growth in vivo, even though significant titers of IFN-alpha (greater than 3,000 IRU/ml) were found in the serum shortly after treatment. Mismatched dsRNA did not induce the production of human IFNs by the tumor cells in vitro. Assays of mouse IFN induction and their in vitro antigrowth effects indicated that the in vivo antiproliferative effect of mismatched dsRNA was probably not due to potentiation of any direct effects by the induced mouse IFNs. Tumor growth inhibition appeared to occur, at least in part, from the significant augmentation (p less than 0.01) of natural killer cell activity by mismatched dsRNA, as measured in the spleen cells of treated mice. These results suggest that, although both IFN-alpha and mismatched dsRNA can be directly antiproliferative against this tumor, either the IFN-independent antitumor effects of mismatched dsRNA or the mismatched dsRNA-induced augmentation of the host immune response plays a major role in tumor regression. Potentially, both mechanisms may be important in this system.

Animals↗
PubMed1983

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro.

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.

Adenine Nucleotides↗
PubMed1995

The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity.

Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.

Amino Acid Sequence↗
PubMed1994

Characterization of the interactions between double-stranded RNA and the double-stranded RNA binding domain of the interferon induced protein kinase.

The interferon-inducible protein kinase, PKR, requires double-stranded (ds) RNA for its activation. We have previously mapped its dsRNA-binding domain (DRBD) to the amino terminal 170 residues (Patel and Sen, 1992). In the present study, we have characterized in detail the interactions between dsRNA and DRBD. For this purpose, DRBD was produced in bacteria as a polyhistidine-tagged protein and purified by affinity chromatography. A polyclonal antibody was raised against purified DRBD. For studying dsRNA-DRBD interactions, a Northwestern assay and an electrophoretic mobility shift assay (EMSA) using a radiolabeled in vitro transcribed 82 bp dsRNA probe was developed. The antiserum reacted with both DRBD and PKR but did not prevent their interactions with dsRNA. DRBD, on the other hand, blocked the activation of PKR by dsRNA. DRBD and the dsRNA probe formed multimeric complexes which were separable by EMSA. The antibody could interact with these complexes and supershift their mobility. Competition with unlabeled dsRNA revealed that the dimeric DRBD-dsRNA complex was much more stable than the monomeric complex. Similar competition assays using 11 different synthetic and natural RNA molecules revealed that only authentic dsRNA molecules could effectively compete with the probe for binding DRBD in a sequence-independent fashion.

Amino Acid Sequence↗
PubMed2026

Development of a Droplet-Based RNA Interference Feeding Assay for Neonates of the Citrus Root Weevil Diaprepes abbreviatus.

The citrus root weevil, Diaprepes abbreviatus, is an economically important pest of citrus and ornamental crops whose subterranean larval feeding damages roots and predisposes plants to secondary pathogen infection. Development of efficient RNA interference (RNAi) delivery methods for early larval stages is essential for functional genomics studies and the evaluation of RNAi-based pest management strategies. In this study, we developed a droplet-based feeding assay for oral delivery of double-stranded RNA (dsRNA) to neonates of D. abbreviatus using chitin synthase 2 (DaCHS2) as a model RNAi target to validate the assay. Feeding solutions containing dsRNA were supplemented with sucrose and bromophenol blue dye, with bromophenol blue used to visually confirm ingestion. Across three independent biological replicates, all neonates exposed to DaCHS2-dsRNA, GFP-dsRNA, and water control droplets were confirmed to have ingested the feeding solution (45/45 neonates per treatment; 100% feeding success). Oral delivery of dsRNA targeting DaCHS2 reduced transcript abundance and was associated with developmental abnormalities and mortality, including incomplete molting, abnormal pigmentation, cuticular deformities, defective pupation, and malformed adults. Regression analysis demonstrated moderate and significant relationship between dsRNA concentration and neonate mortality and developmental abnormalities. RT-qPCR further confirmed reduced DaCHS2 transcript abundance following oral dsRNA exposure. The developed assay provides a simple, reproducible, and minimally invasive proof-of-concept platform for oral dsRNA delivery to D. abbreviatus neonates. The assay requires only small dsRNA volumes, provides visual confirmation of ingestion, and may facilitate laboratory-based screening of additional RNAi target genes in D. abbreviatus and other coleopteran pests.

Animals↗
PubMed1983

Regulation of the antiviral and anticellular activities of interferon by exogenous double-stranded RNA.

The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation of dsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5' oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique.

2',5'-Oligoadenylate Synthetase↗
PubMed2004

Noncatalytic assembly of ribonuclease III with double-stranded RNA.

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.

Amino Acid Sequence↗
PubMed2003

Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts.

Direct assessments of gene function in parasitic flatworms have been hampered by the lack of effective tools to alter gene expression. The aim of the present study was to use RNA-interference (RNAi) to achieve targeted gene knockdown in larval stages of the human blood fluke, Schistosoma mansoni. We selected two S. mansoni genes for RNAi experiments: SGTP1, a facilitated diffusion glucose transporter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When S. mansoni larvae were treated in vitro for 6 days with dsRNA specific to one of these two genes, targeted transcript levels were reduced by 70-80% as determined by quantitative PCR (qPCR), while non-targeted transcripts were unaffected. Parasite exposure to SGTP1 dsRNA, but not GAPDH dsRNA, reduced larval glucose-uptake capacity by 40%, demonstrating that SGTP1 transcript knockdown results in the functional phenotype of reduced glucose transport activity. The effect of dsRNA treatment on transcript level was evident for up to 28 days after an initial dsRNA treatment. Interestingly, dsRNA treatment was effective only when miracidia were allowed to undergo the transition to sporocysts in its presence, while treatment of fully transformed sporocysts was ineffective. Fluorescence patterns in larvae exposed to rhodamine-labeled dsRNA as miracidia and sporocysts were similar, suggesting that the difference in susceptibility to dsRNA treatment between the two life stages may not be due to differences in dsRNA entry. Overall, this technology will enable direct assessment of the roles of individual genes in physiological processes of larval stages of S. mansoni, a crucial step in the identification of novel intervention targets for this important human pathogen.

Animals↗
PubMed1986

In vitro L-A double-stranded RNA synthesis in virus-like particles from Saccharomyces cerevisiae.

Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs). These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity. This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells. The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand. All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute. The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s). We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.

Genes, Fungal↗
PubMed2001

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

Amino Acid Sequence↗
PubMed2001

Role of interferon regulatory factor-1 in double-stranded RNA-induced iNOS expression by mouse islets.

Environmental factors, such as viral infection, have been implicated as potential triggering events leading to the initial destruction of pancreatic beta cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, has been shown in combination with interferon-gamma (IFN-gamma) to stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide production and to inhibit beta cell function. Interferon regulatory factor-1 (IRF-1), the activation of which is induced by dsRNA, viral infection, and IFN-gamma, regulates the expression of many antiviral proteins, including PKR, type I IFN, and iNOS. In this study, we show that IRF-1 is not required for dsRNA + IFN-gamma-stimulated iNOS expression and nitric oxide production by mouse islets. In contrast to islets, dsRNA + IFN-gamma fails to induce iNOS expression or nitric oxide production by macrophages isolated from IRF-1(-/-) mice; however, dsRNA + IFN-gamma induces similar levels of IL-1 release by macrophages isolated from both IRF-1(-/-) and IRF-1(+/+) mice. Importantly, we show that dsRNA- or dsRNA + IFN-gamma-stimulated IRF-1 expression by mouse islets and peritoneal macrophages is independent of PKR. These results indicate that IRF-1 is required for dsRNA + IFN-gamma-induced iNOS expression and nitric oxide production by mouse peritoneal macrophages but not by mouse islets. These findings suggest that dsRNA + IFN-gamma stimulates iNOS expression by two distinct PKR-independent mechanisms; one that is IRF-1-dependent in macrophages and another that is IRF-1-independent in islets.

Animals↗
PubMed2000

Modulation of double-stranded RNA-mediated gene induction by interferon in human umbilical vein endothelial cells.

Endothelial cells respond to double-stranded RNA (dsRNA) with expression of a number of important immunomodulatory and inflammatory response genes, including adhesion molecules, cytokines, and antiviral genes. Considerable differences are seen when genes are induced by dsRNA compared with cytokines. Much higher levels of mRNA for interleukin-6 (IL-6), 2',5'-oligoadenylate synthetase (2',5'-OAS), protein kinase (PKR), and interferon (IFN) regulatory factor-1 (IRF-1) result from incubation with dsRNA than with IL-1beta, tumor necrosis factor-alpha (TNF-alpha), or IFN-alpha, whereas the differences in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin mRNA expression in response to dsRNA, IL-1beta, and TNF-alpha are relatively minor. IFN-alpha priming enhances responsiveness of some, but not all, genes to dsRNA but not to IL-1beta, but the optimal time for pretreatment varies considerably among different dsRNA-responsive genes. Protein translation is reduced in human umbilical vein endothelial cells (HUVEC) in response to incubation with dsRNA, and this decrease is accentuated if cells are primed with IFN-alpha. Despite this decrease, IFN-alpha priming causes very high levels of IL-6 protein expression in response to dsRNA but not in response to IL-1beta or TNF-alpha. These studies demonstrate that priming with class I IFN can enhance the response to dsRNA through the heightened expression of genes that contribute to both the cellular response to viral infection and the host immunologic response.

2',5'-Oligoadenylate Synthetase↗