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At least 91 records · Page 5Linked to original sources
PubMed1992

Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells.

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.

3T3 Cells↗
PubMed2004

[Effects of the dsRNA interferon inducer on the interaction between macrophages and Mycobacterium tuberculosis in vitro].

Effects of the IFN inducer, yeast dsRNA, produced on the interaction between mouse macrophages and phagocytized mycobacteria were experimentally studied in vitro. Mycobacteria were shown to reproduce in macrophages in their initial infection at a ratio of 1:1.25, 1:2.5, 1:5 and 1:10, which was confirmed by an increased insertion of 5.6-[3H]-uracil in M. tuberculosis H37Rv; they also had a destructive impact on macrophages as verified by a higher release of lactic dehydrogenase (LDG) from macrophages. The dsRNA preparation, 40.0, 80.0 and 120.0 microg/ml, was demonstrated to decrease the insertion amount of labeled uracil in phagocytized mycobacteria at a macrophage:mycobacteria ratio of 1:10 and 1:100. The effect depended on a preparation dose and infection degree of macrophages. A decreased release of specific LDG from infected macrophages was shown under the same conditions. The dsRNA affects the interplay of macrophages and phagocytized mycobacteria through inhibiting the vitality of intracellular mycobacteria and through enhancing the stability of macrophages. Special studies denoted that dsRNA, 40.0 microg/ml and 120 microg/ml, activated the production of peroxidation compounds by neutrophils, which phagocytized the sheep erythrocytes. Finally, a possible mechanism of dsRNA impact on the interaction between macrophages and mycobacteria phagocytized by them is under discussion.

Animals↗
PubMed1983

Interferon induction by viruses: one molecule of dsRNA as the threshold for interferon induction.

The studies chronicled in this chapter were chosen largely because they contained data amenable to the quantitative analysis of interferon induction dose-response curves and the IFP activity they represented. The interpretation of the data was predicated on the assumption that a single molecule of dsRNA, when properly introduced into a cell, either as a preformed entity or formed therein following some synthetic event, can induce a quantum yield of interferon. This novel view of interferon induction by viruses has provided an explanation for many seemingly discordant results and offers a unifying hypothesis regarding the nature of the interferon inducer moiety for viruses from widely different families. If we note the reluctance of some to accept dsRNA as a common interferon inducer molecule ( McKimm and Rapp , 1977; Kowal and Youngner , 1978; Joklik , 1980) but recognize that the threshold for activating the interferon induction system is one molecule per cell, many aspects of interferon induction heretofore enigmatic are rendered offerpretable . Furthermore, one molecule of dsRNA per cell suffices to induce a quantum yield of interferon, an apparent expression of the "one-shot affair" of interferon production recognized by Ho (1964). In some cases of induction (the r = 1 type dose-response curve) there is an exquisitely responsive modulation of production when a second molecule of dsRNA is simultaneously introduced into the cell. The experimental approach and concepts discussed herein offer a new perspective on the mechanism of interferon induction by viruses and its regulation, and point out the incredible biological potency of a dsRNA molecule--a molecule found to play a key role in viral infection and host defence (Carter and De Clercq , 1974), regulation of the immune system (Johnson, 1980), and perhaps some yet to be defined function in cell growth (Taylor- Papadimitriou , 1980) and differentiation ( Grossberg and Sabran , 1981-2) through its capacity to activate the interferon system.

Animals↗
PubMed1999

A symmetry mismatch at the site of RNA packaging in the polymerase complex of dsRNA bacteriophage phi6.

The polymerase complex of the enveloped double-stranded RNA (dsRNA) bacteriophage phi6 fulfils a similar function to those of other dsRNA viruses such as Reoviridae. The phi6 complex comprises protein P1, which forms the shell, and proteins P2, P4 and P7, which are involved in RNA synthesis and packaging. Icosahedral reconstructions from cryo-electron micrographs of recombinant polymerase particles revealed a clear dodecahedral shell and weaker satellites. Difference imaging demonstrated that these weak satellites were the sites of P4 and P2 within the complex. The structure determined by icosahedral reconstruction was used as an initial model in an iterative reconstruction technique to examine the departures from icosahedral symmetry. This approach showed that P4 and P2 contribute to structures at the 5-fold positions of the icosahedral P1 shell which lack 5-fold symmetry and appear in variable orientations. Reconstruction of isolated recombinant P4 showed that it was a hexamer with a size and shape matching the satellite. Symmetry mismatch between the satellites and the shell could play a role in RNA packaging akin to that of the portal vertex of dsDNA phages in DNA packaging. This is the first example of dsRNA virus in which the structure of the polymerase complex has been determined without the assumption of icosahedral symmetry. Our result with phi6 illustrates the symmetry mismatch which may occur at the sites of RNA packaging in other dsRNA viruses such as members of the Reoviridae.

Bacteriophage phi 6↗
PubMed1980

The killer phenomenon in Ustilago: electron microscopy of the dsRNA encapsidated in individual virus particles.

From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12-14 X 10(6) daltons of dsRNA. Since the largest extracted molecule is 3.2 X 10(6) D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.

Basidiomycota↗
PubMed1989

Inhibition of HIV-1 proviral DNA synthesis and RNA accumulation by mismatched dsRNA.

The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.

Antiviral Agents↗
PubMed1985

Terminal sequences of the bacteriophage phi 6 segmented dsRNA genome and its messenger RNAs.

The ends of the three dsRNA genome segments (L, M, and S) of bacteriophage phi 6 (strand separated and/or intact) and the 5' ends of the middle and small single-strand messenger RNAs have been sequenced by base-specific partial enzymatic digestion. Terminal sequences for the large and middle dsRNA strands extend about 60 bases. The three dsRNA segments have 18 homologous bases at the left end except for position 2, which differs in the L segment. A 17-base homology defines the right ends of L and M dsRNAs and probably S dsRNA as well. The 5' ends of middle and small messenger RNAs are identical to the corresponding viral (+) strands.

Bacteriophages↗
PubMed1982

Murine immunosuppression with mycoviral dsRNA.

The effect of three different size molecular weight species of mycoviral dsRNA on the immune response to sRBC was tested in C57Bl/6 mice. The various dsRNA species were extracted from electrophoresis polyacrylamide-agarose slab gels. Their molecular weights ranged from 1.0 x 10(6) daltons to 3.5 x 10(6) daltons. All three sizes of mycoviral dsRNA significantly (p less than 0.0001) suppressed the hemolytic antibody titer of mice 8 days after immunizations with 15 micrograms dsRNA/mouse and 10(8) sRBC when compared to control mice which received only sRBC. No immune suppression was observed in any of the mice challenged with a second sRBC immunization 60 days after the first inoculations. Hemagglutination titers at this time were typical of a secondary antibody response to sRBC. In conclusion these three molecular weight mycoviral dsRNA species appeared to be potent immunosuppressors when approximately 15 micrograms/mouse were used.

Adjuvants, Immunologic↗
PubMed1989

Semiconservative strand-displacement transcription of the M2 dsRNA segment of Ustilago maydis virus.

The P1 strain of the Ustilago maydis virus (UmV) is a segmented dsRNA virus with segments designated H1, H2, M1, M2, and L. Incubation of purified virus with a mixture of nucleotides containing 32P-UTP resulted in labeled dsRNA which was retained in the capsid and labeled ssRNA which was released from the capsid. This in vitro transcription reaction was dependent on Mg2+ ion and the optimum concentration for maximum incorporation was 10 mM. The pH and temperature optima were 8.0 and 30 degrees C, respectively. The ssRNA transcripts were precipitated from the supernatant solution of the reaction mixture after ultracentrifugation to separate the virus. Transcription products from supernatant solution hybridized with all five virion dsRNAs. Further studies of the M2 segment indicated that it was labeled within 2 h and the label was completely chased out in 2 h. Analysis of the labeled M2 dsRNA segment by strand-separation gel showed that only one strand (slow moving) was labeled. When both strands were tested in an in vitro translation system, only the slow-moving strand was translated to produce a 24 kDa product. Thus the M2 dsRNA segment of UmV P1 transcribes by a semiconservative strand-displacement mechanism.

Basidiomycota↗
PubMed1994

Endogenous inhibitors of the dsRNA-dependent eIF-2 alpha protein kinase PKR in normal and ras-transformed cells.

The serine/threonine kinase PKR is activated by autophosphorylation in response to nanomolar concentrations of double-stranded RNA and other polyanions. We have previously shown that expression of an oncogenic ras gene induces an endogenous protein inhibitor of PKR activation in murine fibroblasts, as measured by a greatly reduced ability of PKR to autophosphorylate in response to double-stranded RNA in lysates from these ras-expressing cells. Immunoprecipitation of PKR away from the ras-transformed cell lysate restored the ability of PKR to become autophosphorylated. However, the autophosphorylation was no longer dsRNA-dependent. In the present work, PKR immobilized either by immunoprecipitation or by affinity precipitation on Agpoly(I) poly(C) was found to autophosphorylate in a dsRNA-independent manner when incubated in the presence of a detergent lysis buffer and ATP. When lysis buffer was replaced by cytoplasmic extract from normal or ras-transformed cells, autophosphorylation of the immobilized PKR was inhibited in the presence or absence of dsRNA, even though it could be shown that PKR remained bound and intact in the precipitate, and able to autophosphorylate if rewashed with lysis buffer. These findings suggest that PKR activation is regulated by an endogenous inhibitor in murine fibroblasts as well as by dsRNA or other polyanions.

Animals↗
PubMed2005

dsRNA formed as an intermediate during Coxsackievirus infection does not induce NO production in a beta-cell line with or without addition of IFN-gamma.

Virus infection is one environmental factor that has been implicated as a precipitating event initiating beta-cell damage during the development of type 1 diabetes. One aim of this study was to investigate how permissive an insulin-producing beta-cell line, RINm5F, is to enterovirus (EV) infections. A second aim was to study if the viral replicative intermediate, double-stranded RNA (dsRNA), together with IFN-gamma results in nitric oxide (NO) production. Monolayer cultures of RINm5F cells were not permissive to infection with seven different strains of EV. However, when the growth pattern of the beta-cell line changed and the cells started to grow as free-floating RIN cell clusters (RCC), all EV strains replicated. Immunostaining for the Coxsackie-adenovirus-receptor (CAR) detected the protein on the free-floating RIN cell clusters, but not on the RINm5F cells cultured as a monolayer of beta-cells. This shows that the CAR expression can change and/or the CAR protein can be redistributed on the cell surface as a consequence of altered growth pattern thus allowing viral replication in a previously non-permissive beta-cell line. As expected, NO production was significantly increased (p<0.05) by addition of synthetic dsRNA and IFN-gamma to the RCC. In contrast, the dsRNA formed during virus infection with a Coxsackievirus B4 strain (E2) with or without addition of IFN-gamma did not induce NO production in these cells. This indicates that synthetic dsRNA does not mimic a real viral infection in that respect, and suggests an NO-independent mechanism for virus-induced beta-cell damage.

Animals↗
PubMed2004

Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells.

We have used cationic oligopeptide polyarginine-12mer (POA) to deliver double-stranded RNA (dsRNA), prepared in vitro, to tobacco (Nicotiana tabacum) suspension cells. POA interacts electrostatically with dsRNA to form a complex. When dsRNA for the GUS or NPTII gene was delivered into cells carrying the same genes, the corresponding mRNA was degraded. Using RNase protection assay we were able to detect 21-bp small interfering RNA in dsRNA/POA-treated cells. These results demonstrate that POA can be used to deliver dsRNA to induce post-transcriptional gene silencing in plant cells.

Blotting, Northern↗
PubMed2002

Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA.

Interferons enhance the cellular antiviral response by inducing expression of protective proteins. Many of these proteins are activated by dsRNA, a typical by-product of viral infection. Here we show that type-I and type-II interferons can sensitize cells to dsRNA-induced cytotoxicity. In caspase-8- or FADD-deficient Jurkat cells dsRNA induces necrosis, instead of apoptosis. In L929sA cells dsRNA-induced necrosis involves high reactive oxygen species production. The antioxidant butylated hydroxyanisole protects cells from necrosis, but shifts the response to apoptosis. Treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone or overexpression of Bcl-2 prevent this shift and promote necrosis. Our results suggest that a single stimulus can initiate different death-signaling pathways, leading to either necrotic or apoptotic cell death. Inhibition of key events in these signaling pathways, such as caspase activation, cytochrome c release or mitochondrial reactive oxygen species production, tips the balance between necrosis and apoptosis, leading to dominance of one of these death programs.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Base Sequence↗
PubMed2006

Molecular basis for PKR activation by PACT or dsRNA.

The mammalian protein kinase PKR is a critical component of the innate immune response against virus infection. Its cellular actions are mediated by modulating cell signaling and translational regulation. To be enzymatically active, latent PKR needs to be activated by binding to one of its activators, dsRNA or PACT protein. Although the structures of the N-terminal dsRNA-binding domain and the C-terminal kinase domain of PKR have been separately determined, the mode of activation of the enzyme remains unknown. To address this problem, we used biochemical, genetic, and NMR analyses to identify the PACT-binding motif (PBM) located in the kinase domain and demonstrated an intramolecular interaction between PBM and dsRNA-binding domain. This interaction is responsible for keeping PKR in an inactive conformation, because its disruption by point mutations of appropriate residues produced constitutively active PKR. Furthermore, a short decoy peptide, representing PBM, was able to activate PKR by interfering with the intramolecular interaction. These observations suggest a model for PKR activation upon binding of dsRNA or PACT.

Amino Acid Motifs↗
PubMed2006

Inhibition of dsRNA-induced signaling in hepatitis C virus-infected cells by NS3 protease-dependent and -independent mechanisms.

The recent establishment of a robust hepatitis C virus (HCV) cell culture system permits analysis of virus-host interactions during HCV infection. Here, we report that HCV genotype 2a (JFH-1) infection fails to induce IFN-beta or IFN-stimulated gene expression in Huh-7 cells, and that it blocks IFN-beta and IFN-stimulated gene production after transfection of synthetic dsRNA. Overexpression of individual components of the dsRNA-signaling pathway in HCV-infected and uninfected cells indicates that HCV inhibits IFN-beta promoter activity by inactivating the mitochondrial antiviral signaling protein/IFN-beta promoter stimulator 1 (MAVS/IPS-1), while leaving the IFN-induced Janus kinases-signal transducers and activators of transcription (JAK-STAT) signaling pathway intact. We also show that MAVS/IPS-1-dependent IFN-beta promoter activity in HCV-infected cells is fully restored by the nonstructural protein 3 (NS3) protease inhibitor BILN2061. In contrast, synthetic dsRNA-induced IFN-beta promoter activity is not restored by BILN2061, although it is partially restored by overexpression of RIG-I. These results support recently reported evidence that the HCV NS3 protease blunts the ability of HCV to induce IFN-beta promoter activity by proteolytically cleaving MAVS/IPS-1. The results also suggest that HCV blocks the synthetic dsRNA-induced signaling pathway at a point upstream of MAVS/IPS-1, and that it does so by an NS3-independent mechanism.

Active Transport, Cell Nucleus↗
PubMed2002

The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR.

TRBP (HIV-1 transactivating response (TAR) RNA-binding protein) and PKR, the interferon-induced dsRNA-regulated protein kinase, contain two dsRNA binding domains. They both bind to HIV-1 TAR RNAs through different sites. Binding to dsRNA activates PKR that phosphorylates the eukaryotic initiation factor eIF-2alpha leading to protein synthesis inhibition. TRBP and PKR can heterodimerize, which inhibits the kinase function of PKR and has a positive effect on HIV-1 expression. In this study, an in vitro reticulocyte assay revealed the poor expression of TAR containing CAT RNAs compared with CAT RNAs. Addition of TRBP restored translation efficiency of TAR-CAT RNA and decreased the phosphorylation status of eIF-2alpha, confirming its role as a PKR inhibitor. Unexpectedly, eIF-2alpha was phosphorylated in the presence of TAR-CAT as well as CAT RNA devoid of the TAR structure. TRBP inhibited eIF-2alpha phosphorylation in both cases, suggesting that it restores the translation of TAR-CAT RNA independently and in addition to its ability to inhibit PKR. TRBP activity on gene expression was then analyzed in a PKR-free environment using PKR-deficient murine embryo fibroblasts. In a transient reporter gene assay, TRBP stimulated the expression of a TAR-containing luciferase 3.8-fold whereas the reporter gene with mutated TAR structures or devoid of TAR was stimulated 1.5- to 2.4-fold. Overall, the activity of TRBP2 was higher when the 5'-end of the mRNA was structured and was mediated independently by each dsRBD in TRBP. Increasing concentrations of TRBP showed no significant modification of the luciferase RNA levels, suggesting that TRBP stimulates translation of TAR-containing RNAs. Therefore, TRBP is an important cellular factor for efficient translation of dsRNA containing transcripts, both by inhibiting PKR and in a PKR-independent pathway.

Animals↗
PubMed1991

Effect of dsRNA from phi 6 bacteriophage on herpetic infection in cell culture and an animal model.

The double-stranded (ds) RNA from phi 6 bacteriophage inhibited herpes simplex virus type 1 (HSV-1) and HSV-2 infection in MA-104 cells but not in Vero cells. HSV-2 was more sensitive to this effect than HSV-1, with the HSV-2 ED50 being 0.25 micrograms/ml and the HSV-1 ED50 1.68 micrograms/ml. On genital infection by HSV-2 in guinea pigs, phi 6 dsRNA was more effective by intravaginal (P less than 0.05) than by intraperitoneal administration. A single dose of dsRNA of 600 micrograms/kg by intravaginal route modified favorably the natural course of the genital herpes in the treated animals (p less than 0.001). Compared with the infected controls, they showed a faster recovery with better healing of lesions; and the number and severity of recurrence was low. No mortality was observed and the control infected animals showed a mortality of 39%. Sera from dsRNA-treated animals showed antiviral activity with a 50% plaque-depressing dose (PDD50) of 10(1.5)/150 microliters; no antiviral activity was found in sera either from control infected or uninfected animals. No adverse effect was observed on the rate of growth of uninfected dsRNA-treated controls.

Animals↗