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At least 739 records · Page 41Linked to original sources
PubMed1995

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-I LTR.

We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

3T3 Cells↗
PubMed1997

Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene.

This work investigated whether the Escherichia coli RNase III gene, rnc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene. Like E3L, rnc+ codes for a dsRNA binding protein that contains an additional nucleolytic activity. Rnc genes were cloned into the eukaryotic expression vector pMTVa-, expressed in COS-1 cells, and shown to be functional. Transient rescue experiments in HeLa cells demonstrated that the cleavage function of the rnc+ gene was necessary for full rescue of vp1080. The rnc 70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. The rnc 105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. The rnc genes were also inserted into the E3L locus of vp1080. While recombinants containing the rnc+ gene or the rnc 70 gene regained the IFN resistance phenotype in RK13 cells, full host range of vaccinia virus was only restored in the recombinant containing the rnc+ gene. Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype. The vp-rnc 105 recombinant behaved similarly to vp1080.

Amino Acid Sequence↗
PubMed2002

Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus.

The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.

Amino Acid Sequence↗
PubMed1993

Enigmatic double-stranded RNA in Japonica rice.

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12,351 nucleotides long and encoded more than 4,117 amino acid residues.

Base Sequence↗
PubMed1979

The regulation of RNA synthesis in yeast IV. Synthesis of double-stranded RNA.

A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.

Cell Division↗
PubMed1991

Sequences of three dsRNAs associated with La France disease of the cultivated mushroom (Agaricus bisporus).

La France disease of the cultivated mushroom, Agaricus bisporus, is known to be associated with the presence of a number of dsRNA segments. The nucleotide sequences of the dsRNAs M2 (1.3 kb), M1 (1.55 kb) and L3 (2.8 kb), invariably associated with the disease, were determined. Putative coding sequences for proteins with molecular weights of 38, 40 and 87 kDa were found for M2, M1 and L3 dsRNAs, respectively. The average G + C content of these dsRNAs was 43%, close to that of A. bisporus nuclear DNA. The nucleotide sequences, as well as the amino acid sequences, appear to be unique, as no matching sequences could be found among databases. S3 dsRNA (0.39 kb), which is occasionally found in large amounts in diseased mushrooms, is an internally deleted variant of M2 dsRNA and is largely composed of the non-coding ends of that dsRNA.

Agaricus↗
PubMed1989

A very small viral double-stranded RNA.

UmV is a double-stranded RNA (dsRNA) virus of the corn fungal pathogen Ustilago maydis. UmV has no infectious cycle. Some UmV subtypes have viral dsRNAs encoding secreted toxins that kill sensitive cells of the same species and related species. There are three viral subtypes, P1, P4 and P6, which differ in the specificity of their secreted killer toxins. Each has three size classes of dsRNA: H (heavy), M (medium) and L (light). The L segments of UmV are unique in being derived from one end of the larger M segments. We have sequenced P1 L and placed it at the 3' end of the P1 M1 plus strand. In their overlapping regions, these dsRNAs are identical in sequence. In vitro translation of P1 M1 results in a peptide whose size is consistent with its being encoded by the non-L region of M1. P1 L is a very small dsRNA of 355 bp. It has no long open reading frames and produces no detectable in vitro translation product. The sequence of P1 L suggests that it is derived by a process unique among dsRNA viruses: replication and packaging of the 3' end fragment of a processed mRNA.

Base Sequence↗
PubMed1984

Anti-DNA.RNA sera. Specificity tests and application in quantitative in situ hybridization.

The specificity of anti-nucleic acid antisera can immunocytochemically be evaluated with test systems which apply various nucleic acids immobilized to inert matrices. When using polyrA.polyrU as a model compound for dsRNA, it is important to prevent the formation of the triple stranded form polyrA.(polyrU)2. Anti-dsRNA antibodies which, when tested with the correct test system, proved to be present in an earlier described anti-DNA.RNA serum, could be removed by adsorption. By cytofluorometric comparison of the immuno-fluorescence signals obtained with the anti-dsRNA containing serum and the absorbed serum, it could be shown that the anti-dsRNA antibodies do not contribute to the specific signals measured after in situ hybridization. Repetitive incubations with the anti-DNA.RNA serum led to a considerable increase in immunofluorescence signal.

Animals↗
PubMed1999

Double-stranded RNA in the entomopathogenic fungus metarhizium flavoviride.

Bands of dsRNA were detected in five out of seven isolates of the entomopathogenic fungus Metarhizium flavoviride. The identity of these bands was proven by RNase and S1-nuclease treatments. The transference of dsRNA between isolates (from CG291 to CG442) was successfully carried out through forced heterokaryons. Isogenic strains, with or without dsRNA, were submitted to virulence tests against the grasshopper Rhammatocerus schistocercoides. In contrast to what has been found in some phytopathogenic fungi, these dsRNA fragments did not cause hypovirulence to M. flavoviride.

Animals↗
PubMed2006

A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production.

Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanism. However, a strict comparison of TGS and PTGS has been difficult to achieve because it generally relies on the analysis of distinct silencing loci. We describe a single transgene locus that triggers both TGS and PTGS, owing to the production of dsRNA corresponding to promoter and transcribed sequences of different target genes. We describe mutants and epigenetic variants derived from this locus and propose a model for the production of dsRNA. Also, we show that PTGS, but not TGS, is graft-transmissible, which together with the sensitivity of PTGS, but not TGS, to RNA viruses that replicate in the cytoplasm, suggest that the nuclear compartmentalization of TGS is responsible for cell-autonomy. In contrast, we contribute local and systemic trafficking of silencing signals and sensitivity to viruses to the cytoplasmic steps of PTGS and to amplification steps that require high levels of target mRNAs.

Epistasis, Genetic↗
PubMed2003

A simple "soaking method" for RNA interference in the planarian Dugesia japonica.

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.

Animals↗
PubMed2004

RNA interference: the molecular immune system.

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

AIDS Vaccines↗
PubMed2006

Bluetongue virus and double-stranded RNA increase human vascular permeability: role of p38 MAPK.

Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in trans-endothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases.

Bluetongue virus↗
PubMed2004

The gene product specified by a double stranded RNA in the flax rust (Melampsora lini) is expressed during rust growth.

Strain SP6 of the flax rust (Melampsora lini) contains 11 double-stranded RNAs (dsRNAs) of unknown function. A large open-reading frame (B3ORF1 in dsRNA B3 encodes a polypeptide of 614 amino acids, and using an antiserum raised against the B3ORF1-glutathione S-transferase fusion protein prepared from a bacterial expression system, we have detected the presence of a 67 kDa polypeptide in rust urediospores. This polypeptide, identical in size to that of the predicted translation product of B3ORF1 , was not detected in spores from either a fungal strain lacking the B3 dsRNA or an isogenic strain containing no dsRNA. These data indicate that B3ORF1 present in the flax rust B3 dsRNA is expressed in vivo which warrants farther investigation in search for its function during rust development.

Basidiomycota↗
PubMed1986

The internal polyadenylate tract of yeast killer virus M1 double-stranded RNA is variable in length.

The 1.8-kbp M1 double-stranded (ds) RNA from type 1 killer strains of Saccharomyces cerevisiae contains an internal 200-bp adenine- and uracil-rich region. We have previously demonstrated that this region consists primarily of adenine residues on the plus strand of M1 dsRNA and on the full-length, in vitro synthesized (+) transcript (denoted m) of M1 dsRNA, neither of which contains 3'-terminal polyadenylate. We now show that there is variability in the length of the polyadenylate tracts of m transcripts synthesized in vitro by virions purified from either of the K1 diploid killer strains A364A X S7 or A364A X 1384. This variability reflects size differences seen in the corresponding M1 dsRNA genomes which, along with other data presented, localizes the variability in the length of M1 dsRNA to the adenine- and uracil-rich region.

Molecular Weight↗
PubMed1987

The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein.

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.

Infectious bursal disease virus↗
PubMed1987

Association of reovirus proteins with the structural matrix of infected cells.

The interaction of reovirus with the cytoskeleton was investigated. The soluble components of infected cells were extracted with the nonionic detergent NP-40 in a physiological buffer, and a cytoskeletal extract was prepared from the detergent-insoluble fraction. We observed a selective association of viral-specified products with the cytoskeleton that was temporally controlled. Viral dsRNA appeared first on the framework but after several hours was found also in the soluble phase, encapsidated in mature virions. The initial viral translation products were associated exclusively with the soluble fraction, but concomitant with the appearance of dsRNA, viral proteins microNS and sigma 3 were detected on the cytoskeleton. Several hours later, all viral proteins were detected on the framework. Viral polypeptide microNS exhibited unique spatial distribution patterns that correlated with viral assembly: Before dsRNA replication, it appeared as diffusely distributed protein; a few hours later, it was detected in punctate foci interconnected by tiny filaments; several hours later, it appeared as an extensive fiber network that traversed the foci. The other viral proteins were detected only within viral foci. MicroNS remained bound to the matrix fraction after treatment with DNase, Mg2+, and high salt, treatments that released other viral proteins. This distribution pattern was virus-directed because passage of virus at high multiplicity of infection induced mutations that prevented assembly of the microNS-coated filament organization. A small fraction of the viral-specified products that included polypeptide microNS, but not viral dsRNA, was coprecipitated from cytoskeletal extracts with proteins of mol wt approximately 55K by monoclonal antibodies that recognized tubulin and vimentin. Disruption of this interaction by long exposure to colchicine did not prevent association of viral proteins or RNA with the matrix, indicating that viral products were not transported through these interactions. The results indicate that reovirus morphogenesis includes temporal and spatial controls not described previously.

Animals↗
PubMed1988

Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain.

L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first [+] strand then [-] strand), intraviral process. New particles are formed by packaging (+) strands. Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA. We find that the isolated binding complex replicates only if it was formed in the presence of the host factor. The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots. The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA. The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand. Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.

Amino Acid Sequence↗