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PubMed1992

The role of double-stranded RNA in induction of the acute-phase response in an abortive influenza virus infection model.

The inducer of the acute-phase response in "flu-like" viral infections is not defined. The hypothesis that virus-associated double-stranded (ds) RNA serves this function was investigated by comparison of several acute-phase responses (fever and sleep patterns, white and nucleated red blood cell levels, serum antiviral activity and ceruloplasmin) induced by the synthetic dsRNA polyriboinosinic:polyribocytidylic acid (poly[rI:rC]) with those induced by influenza virus in rabbits. The capacity of either dsRNA or influenza virus to induce hyporesponsiveness with respect to these acute-phase parameters upon rechallenge with the same agent or cross-challenge 24 h later was also examined. Poly(rI:rC) induced only minimal hyporesponsiveness to itself but was a potent inducer of hyporesponsiveness to virus with respect to fever, sleep, leukograms, and antiviral activity. Therefore, poly(rI:rC) can substitute for virus in terms of induction of acute-phase hyporesponsiveness, suggesting that dsRNA of viral origin triggers the acute-phase response in this model of influenza.

Acute-Phase Reaction↗
PubMed1990

Double-stranded RNA and bacterial lipopolysaccharide enhance sensitivity to TNF-alpha-mediated cell death.

The effect of double-stranded RNA (dsRNA) and bacterial lipopolysaccharide on the sensitivity to tumor necrosis factor (TNF)-alpha-mediated cell death was studied in an in vitro system. Since secretion of TNF-alpha is a part of the early host response to viral and bacterial infection, we examined whether mimicking the infection with viral and bacterial products could affect the response of cells to TNF-alpha. Incubation of WEHI 164 fibrosarcoma cells with dsRNA or lipopolysaccharide (LPS) significantly increased their sensitivity to TNF-alpha-mediated lysis and to TNF-secreting inflammatory T cell-mediated lysis. Thus, these products could induce increased sensitivity to TNF-alpha in cells in an inflammatory focus, possibly contributing to selective elimination of infected but not healthy cells by this non-specific cytokine. Additionally, our data show that both dsRNA and LPS, as well as TNF-alpha itself, rapidly induce nuclear factor-kappa B (NF-kappa B), a DNA-binding protein implicated in regulation of gene expression. We suggest that NF-kappa B could regulate genes crucial for the induction of cell death by TNF-alpha.

Animals↗
PubMed1983

Two Ustilago maydis viral dsRNAs of different size code for the same product.

UmV is a double-stranded RNA (dsRNA) virus of the corn fungus Ustilago maydis. 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 dsRNAs: H (heavy), M (medium), and L (light). We find that, unique among dsRNA viruses, two segments of different size code for the same product--the toxin resistance factor. The smaller dsRNA (L) is homologous to one end of the larger (M), and may have arisen by replication and packaging of a sub-genomic mRNA. We have also compared all the UmV dsRNAs with each other and with the dsRNAs of the similar yeast virus (ScV) by Northern gel and by 3' sequence analysis. Like those of ScV, many of the UmV dsRNAs have one 3' terminus with the sequence UUUUUCAOH or UUUUUCGOH. The H and L dsRNAs of similar size in different viral subtypes are generally related in sequence. The UmV H dsRNAs of different size are not detectably related in sequence.

Base Sequence↗
PubMed1985

Long internal inverted repeat in a yeast viral double-stranded RNA.

The Saccharomyces cerevisiae viruses are non-infectious double-stranded (ds) RNA viruses present in most laboratory strains of yeast. Their genome consists of one or more dsRNAs separately encapsidated in particles composed mainly of one polypeptide, which has a Mr of 88 kdaltons in the best-studied viral subtype. A large viral dsRNA (L1, of 4.7 kb) encodes the capsid polypeptide. We have determined the sequences of a number of cDNA clones homologous to portions of L1 and mapped them by a novel heteroduplex technique. Several of these clones originate from a region of L1 2.3-2.5 kb from the 5' end of the plus strand that contains stop codons in all three reading frames in the plus strand. We therefore suspect that the capsid polypeptide gene lies in the 5' 2.3-2.6 kb of the plus strand. One of the cloned cDNAs has an inverted repeat of 170 bp that appears to be present in its parental RNA. The inverted repeat in L1 is the longest known inverted repeat in a viral dsRNA and the only known non-terminal inverted repeat. It might serve the function of creating two mRNAs from one viral dsRNA.

Amino Acid Sequence↗
PubMed1990

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

Adenoviruses, Human↗
PubMed1975

Ionic strength effects on the stability and conformation of Penicillium chrysogenum mycophage double stranded RNA.

The effect of [Na+] on the stability and conformation of penicillium chrysogenum mycophage dsRNA (PCMdsRNA) was investigated using CD and UV optical techniques. Thermal melting profiles reveal prominent fine structure attributed to at least four regions of structural dissimilarity. A constant increased thermal stability of the dsRNA compared to DNA of the same base composition was observed over a concentration range of 1.5 times 10- minus 4 M to 4.5 times 10- minus 2 M Na+. At low ionic strengths ([Na+] less than 10- minus 3 M) Tm becomes independent of further decrease in [Na+] unless the dsRNA is exposed to high concentrations of EDTA, suggesting the involvement to tightly bound divalent cation. At relatively high ionic strengths ([Na+] greater than 0.1 M) a postulated A leads to A' ... conformation change occurs.

Circular Dichroism↗
PubMed1992

Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.

Base Sequence↗
PubMed1993

A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one or more noninfectious viruses of a progenitor cell, an origin that predates the differentiation of protozoans and fungi. The cause of this preservation of sequence appears to be constraints placed on the RDRP by the life-style of these viruses--the maintenance of a stable, persistent, noninfectious state.

Amino Acid Sequence↗
PubMed1994

NF-kappa B-independent activation of beta-interferon expression in mouse F9 embryonal carcinoma cells.

We have examined the behaviour of the beta-interferon promoter in mouse F9 embryonal carcinoma cells. In undifferentiated cells, the beta-interferon promoter is not responsive to dsRNA or to Sendai virus. In cells stimulated to differentiate into parietal endoderm by treatment with retinoic acid, the beta-interferon promoter responds to both inducers, but only Sendai virus can activate the transcription factor NF-kappa B previously thought to be essential for beta-interferon induction. Differentiated F9 cells therefore present an unprecedented situation in which induction of the beta-interferon gene does not require NF-kappa B. In addition to these differences, induction by dsRNA, but not by Sendai virus, is significantly enhanced by a pretreatment with interferon (priming). These observations suggest that paramyxo-viruses can participate in beta-interferon induction in a manner that is distinct from a simple generator of dsRNA. Analysis of the promoter requirements for induction in differentiated F9 cells suggests that induction is brought about by a novel mechanism using the currently identified regulatory domains.

Animals↗
PubMed2000

Regulation of the RNA-dependent protein kinase by triple helix formation.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phos-phorylates the alpha-subunit of the translation initiation factor eIF-2, inhibiting its function. PKR is activated in vitro by binding to double-stranded RNA (dsRNA) molecules of approximately 30 bp or longer. Here we show that triple helix forming oligonucleotides (TFOs) inhibit dsRNA binding to the isolated RNA binding domain of PKR. The inhibition is specific to the targeted RNA and dependent on TFO length. Binding to a 30 bp duplex is inhibited by a 28 nt TFO and a 20 nt TFO with an IC(50) of 35 +/- 2 and 210 +/- 22 nM, respectively. An 18 nt TFO partially inhibits binding. The activation of the kinase domain of PKR by a 30 bp RNA duplex is also inhibited by a 28 nt TFO. Inhibition of binding is most effective when the triple helix is formed prior to addition of the protein. These results indicate that triplex formation can be used to prevent the binding of an RNA binding protein with dsRNA-binding motifs.

Base Sequence↗
PubMed2004

Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro.

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.

Cell Cycle Proteins↗
PubMed2005

A computational study of off-target effects of RNA interference.

RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of approximately 21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, 'off-target effects' compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

Animals↗
PubMed2005

Binding of herpes simplex virus-1 US11 to specific RNA sequences.

Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.

Base Sequence↗
PubMed2006

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Binding Sites↗
PubMed2004

RNAi expression mediated inhibition of HCV replication.

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Cell Line, Tumor↗
PubMed2006

Analysis of the interaction between human TLR3 ectodomain and nucleic acids.

Toll-like receptor 3 (TLR3) recognizes dsRNA of viral origin and polyriboinosine-polyribocytidylic acid (poly (I:C)). TLR3 mediates the activation of IRF-3 and NF-kappaB and thereby the secretion of type I interferons and inflammatory cytokines. However, the mechanism of this activation is poorly understood. To study the molecular recognition events and biochemical interactions between TLR3 and dsRNA, human TLR3 ectodomain (ECD) fused with a signal peptide at the N-terminus and 6 x His-tag at the C-terminus (TLR3-ECD) was obtained by the baculovirus expression system. To examine the various nucleic acids binding to TLR3-ECD in vitro, a filter binding assay was carried out at pH 4.2-7.6. Interaction of TLR3-ECD with various nucleic acids (particularly dsRNA, in vitro transcripts of tRNA and HCV NS3 aptamer) required an acidic pH.

Binding Sites↗
PubMed2002

Ingestion of double-stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference.

RNA interference is of value in determining gene function in many organisms. Plant parasitic nematodes are refractory to microinjection as a means of introducing RNA and do not show any oral uptake until they are within plants. We have used octopamine to stimulate uptake by preparasitic second stage juveniles of two cyst nematodes, Heterodera glycines and Globodera pallida. This new technique was used to facilitate uptake of double stranded RNA (dsRNA) together with fluoroscein isothiocyanate as a visual marker. Targeting cysteine proteinases did not reduce the number of parasites but caused a shift from the normal female/male ratio of 3:1 to 1:1 by 14 days postinfection (dpi). Exposure of H. glycines to dsRNA corresponding to a newly characterized protein with homology to C-type lectins did not affect sexual fate, but 41% fewer parasites were recovered from the plants. As expected, treatment with dsRNA corresponding to the major sperm protein (MSP) had no effect on either parasite development or sexual fate over 14 days. Northern analysis showed lower transcript abundance for the two targeted mRNAs that occur in J2, plus a later inhibition for MSP transcripts when males developed sperm at 15 dpi. These findings establish a procedure for RNAi of plant parasitic nematodes.

Animals↗
PubMed2003

Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing.

Specific post-transcriptional gene silencing (PTGS) of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA (dsRNA) molecules. In plants, PTGS is part of a defense mechanism against virus infection. We have previously shown and patented that direct delivery to nontransgenic plants of dsRNA derived from viral sequences specifically interfere with virus infection. Here, we show that transient expression of constructs encoding hairpin RNA homologous to a rapidly replicating plant tobamovirus also interferes with virus multiplication in a sequence-dependent manner. A three-day lag period between delivery of hairpin RNA and virus into the same tissues completely block virus infectivity. Several hallmarks characteristic of PTGS were associated with viral interference mediated by hairpin RNA: high level of sequence identity between the hairpin RNA and the target RNA, presence of siRNAs in extracts derived from leaves infiltrated with hairpin RNA, and helper component-proteinase (HC-Pro) of potyviruses, a suppressor of PTGS, overcame interference. No evidence for a mobile silencing suppression signal induced by transient expression of HC-Pro was observed. The approach described here has the potential to be used as a versatile tool for studying the onset of PTGS in cases involving virus infection, in opposition to dsRNA-transgenic plants, which allow primarily for the study of PTGS maintenance.

Gene Silencing↗