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PubMed1987

The dsRNA of Trichomonas vaginalis is associated with virus-like particles and does not correlate with metronidazole resistance.

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains of Trichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA-positive strains to contain virus-like particles 32-38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist.

Animals↗
PubMed2006

Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA.

Yellow head virus (YHV) is an invertebrate nidovirus which causes a severe mortality in cultured Penaeus monodon. The mortality may be prevented by prior treatment of shrimps with YHV-protease dsRNA. Whether the YHV infected shrimp might be cured by the dsRNA remains to be investigated. P. monodon injected with 10(-6) YHV showed a high virus replication and mortality within 2 days. Injection of 25 microg YHV-protease dsRNA at 3, 6, 12 or 24 h post YHV infection showed a strong inhibition of YHV replication up to 12 h. Unrelated dsRNA-GFP showed no inhibition, indicating that the inhibition was nucleic acid sequence specific through RNAi pathway. Shrimp mortality could be prevented at 3h post YHV infection by the dsRNA, but not at 24 h. These results demonstrate that YHV-protease dsRNA gives therapeutic effect and pave the way to develop a cure for YHV-infected shrimps.

Animals↗
PubMed2004

Complexity of dsRNA mycovirus isolated from Fusarium graminearum.

Fusarium graminearum is the causal agent of a serious scab disease of small grains in Korea. We screened 827 isolates of F. graminearum from diseased barley and maize and tested for the presence of double-stranded RNA (dsRNA) mycovirus. Of them, 19 isolates contained various sizes of dsRNAs. A dsRNA associated with pronounced morphological changes including reduction in mycelial growth, increase in dark orange to red pigmentation, reduced sporulation and virulence was previously observed in nine dsRNA-containing Fusarium isolates (Chu et al., Appl Env Microbiol 68, 2529-2534, 2002). Ten additional isolates were found infected with dsRNA mycoviruses. These mycoviruses contain 2-4 different segments of dsRNAs with the size-range of approximately 1.7-10 kbp in length. The presence of dsRNAs did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100%. Interestingly, dsRNA mycovirus found in F. graminearum isolates, JB33 and JNKY19, that show the pattern of mixed infection of two different viruses were transmitted to all progeny conidia and ascospores. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs.

Amino Acid Sequence↗
PubMed2006

c-Jun N-terminal kinase negatively regulates dsRNA and RSV induction of tumor necrosis factor- alpha transcription in human epithelial cells.

Secretion of inflammatory cytokines is the initial step of the immune response to viral infections. This innate immune response is mediated by the expression of a variety of cytokines, exemplified by tumor necrosis factor- alpha (TNF-alpha). The presence of dsRNA during viral infections is a key step in activation of several signaling pathways, including protein kinase R (PKR), toll-like receptor 3 (TLR3), mitogen-activated protein kinase (MAPK), activator protein-1 (AP-1), interferon regulatory factors (IRFs), and NF-kappaB pathways, which are all relevant in the expression of inflammatory cytokines. We previously reported that PKR and p38 MAPK were required for dsRNA and viral induction of inflammatory cytokines in epithelial cells. Here, we report that activation of c-Jun N-terminal kinase (JNK) during dsRNA treatment or respiratory syncytial viral (RSV) infection negatively regulates the induction of TNF-alpha in human epithelial cells. Inhibition of JNK by a pharmacologic inhibitor showed that expression of TNF-alpha increased following both dsRNA treatment and infection with RSV. Importantly, transfection of epithelial cells with a dominant-negative mutant of JNK significantly increased dsRNA induction of TNF-alpha. The mechanism by which JNK inhibition increases TNF-alpha induction appears to be through p38 MAPK activation. Our data show that JNK is a negative regulator of dsRNA and RSV induction of TNF-alpha expression and, thus, may act as a counterbalance to proinflammatory signals generated during viral infections.

Anthracenes↗
PubMed1983

Yeast killer dsRNA plasmids are transcribed in vivo to produce full and partial-length plus-stranded RNAs.

In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae. Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species. Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs. 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated. Other L-dsRNA gene products and their transcripts may exist. For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA. Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m. A functional model for M1-dsRNA killer plasmid structure is presented.

Animals↗
PubMed1992

A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA.

We have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectira (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12.5 kbp and is polyadenylated at the 3' terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from European isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3'-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5'-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713.

Ascomycota↗
PubMed2004

Baculovirus and dsRNA induce Hemolin, but no antibacterial activity, in Antheraea pernyi.

Hemolin is one of the haemolymph proteins most strongly induced upon bacterial infection in Lepidoptera. When we applied RNA interference (RNAi) to suppress Hemolin expression in the Chinese oak silk moth Antheraea pernyi, we discovered that Hemolin is induced by double-stranded RNA (dsRNA) per se. As dsRNA is recognized as a virus pattern molecule, we then investigated the effect of a baculovirus (ApNPV) infection. We found that Hemolin is induced and expressed with similar kinetics as upon dsRNA injection. Notably, no Attacin gene expression or antibacterial activity was recorded. When baculovirus and high amounts of dsRNA were coinjected, the viral symptoms appeared earlier with Hemolin dsRNA than with GFP dsRNA. This indicates that silencing of hemolin affected the progress of the viral infection.

Animals↗
PubMed2005

Immune responses to dsRNA: implications for gene silencing technologies.

Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.

Animals↗
PubMed2005

CXCR2/CXCR2 ligand biological axis impairs alveologenesis during dsRNA-induced lung inflammation in mice.

The histologic phenotype of bronchopulmonary dysplasia (BPD) is characterized by decreased alveolization and is preceded by infiltration of activated neutrophils into the lung that can lead to sustained lung injury and potential interruption of normal lung development. Potential pathogens triggering early neutrophil influx include either prenatal or postnatal exposure to bacteria or viruses. Specific mechanisms recruiting neutrophils to the lung and subsequently decreasing alveolization during virus-induced lung inflammation and injury have not been fully elucidated. Because CXC chemokines, such as CXCL1 and CXCL2/3 acting through their putative receptor, CXCR2, are potent neutrophil chemoattractants, the authors investigated their role in dsRNA-induced lung injury and decreased alveolization, in which dsRNA (poly IC) is a well-described synthetic agent mimicking acute viral infection. Intratracheal dsRNA led to significant increases in neutrophil infiltration and lung injury at 72 hours and to decreased alveolization at 5 days after dsRNA exposure in newborn (10 days old) BALB/c mice, when compared with controls treated and not treated with ssRNA (poly C). Expression of CXCL1 and CXCR2 paralleled neutrophil recruitment to the lung and preceded the decrease in alveolization. Inhibition of CXCR2/CXCR2 ligand interaction by pretreating dsRNA-exposed mice with an anti-CXCR2 neutralizing antibody significantly attenuated neutrophil sequestration and lung injury, and preserved normal alveolization. These findings demonstrate that the CXCR2/CXCR2 ligand biologic axis plays an important role during the pathogenesis of dsRNA-induced lung injury and decreased alveolization and may be relevant to the pathogenesis of BPD.

Animals↗
PubMed2001

The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism.

The last few years have witnessed the appreciation of dsRNA as a regulator of gene expression, a potential antiviral agent, and a tumor suppressor. However, in spite of these clear effects on the cell function, the mechanism that controls dsRNA maturation and stability remains unknown. Recently, the discovery of eukaryotic orthologues of the bacterial dsRNA specific ribonuclease III (RNase III) suggested a central role for these enzymes in the regulation of dsRNA and eukaryotic RNA metabolism in general. This article reviews the structure-function features of the eukaryotic RNase III family and their roles in dsRNA metabolism with an emphasis on the yeast RNase III. Yeast RNase III is involved in the maturation of the majority of snRNAs, snoRNAs, and rRNA. In addition, perturbation of the expression level of yeast RNase III alters meiosis and causes sterility. These basic functions of the yeast RNase III appear to be widely conserved which makes it a good model to understand the importance of eukaryotic dsRNA metabolism.

Animals↗
PubMed2026

Screening, optimization and artificial recombination of dsRNA fragments for RNAi-mediated pest resistance in Apolygus lucorum.

RNA interference (RNAi) is an eco-friendly strategy for pest management, with double-stranded RNA (dsRNA) as the core functional component. In this study, three RNAi target genes (Ubx, wupA and Dpp) with strong lethal effects on Apolygus lucorum were screened via microinjection. The 7-day cumulative mortalities were 56.67 ± 3.33% for dsUbx, 94.44 ± 1.11% for dswupA and 92.22 ± 1.11% for dsDpp. We optimized dsRNA sequences by removing conserved sequences in non-target organisms based on homology alignment and off-target risk analysis. The optimized fragments dswupA-OTE and dsDpp-OTE still exhibited high insecticidal activity, with 7-day cumulative mortalities of 77.78 ± 2.94% and 70.00 ± 1.93%, respectively. We also evaluated the effects of dsRNA length and target sites on RNAi efficiency and screened potent short dsRNA fragments. Novel artificially recombinant dsRNAs were constructed by assembling effective short fragments from different genes, which retained strong insecticidal activity despite shorter sequence length. This study verifies the feasibility of multi-target recombinant dsRNA for pest control and provides a theoretical basis for developing multi-gene RNAi technologies against A. lucorum.

Apolygus lucorum↗
PubMed1991

Substrate specificity of the dsRNA unwinding/modifying activity.

Double-stranded RNA (dsRNA) unwinding/modifying activity, which is present in a wide range of eukaryotic cells, has been previously shown to convert up to 50% of adenosine residues to inosines within intermolecular dsRNA. In the present study, we report that this activity also modifies, though slightly less efficiently, intramolecular double-stranded regions of synthetic RNAs. Our results widen the range of the possible biological substrates for the activity since many stem and loop type RNA secondary structures (intramolecular dsRNA), present in eukaryotic as well as viral transcripts, can potentially serve as substrates. In addition, we have found that the dsRNA unwinding/modifying activity requires a double-stranded region of at least 15-20 base pairs (bp) for substrate recognition. Furthermore, modification efficiency was found to be critically dependent on the length of the double-stranded region; as the size decreased below 100 bp, it dropped precipitously. Our results suggest that efficient modification may occur only with relatively long (greater than 100 bp) dsRNA, perhaps because multiple copies of the enzyme must be bound.

Base Sequence↗
PubMed2001

RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA.

RNA interference (RNAi), the targeted mRNA degradation by double-stranded RNA (dsRNA), is a useful tool for studying gene function in several organisms. Here we report results of experiments with mammalian dsRNA expression vectors that are suitable to study gene function in mouse oocytes and preimplantation embryos. The plasmid vectors were constructed to contain the SV40 small intron, EGFP coding sequence to permit detection of expression, and an inverted repeat to mos mRNA that would form a hairpin dsRNA. Results of the experiments indicated that (i) hairpin dsRNA was just as effective as dsRNA (i.e., annealed sense and antisense RNA) in promoting the destruction of targeted mRNA, (ii) the EGFP marker could be expressed from the construct, and (iii) the distance of the SV40 intron from the inverted repeat was critical for the transcribed RNA to function in RNAi.

Animals↗
PubMed1997

A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix.

Amino Acid Sequence↗
PubMed2002

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.

Animals↗
PubMed1997

Incomplete dsRNA genomes in purified infectious pancreatic necrosis virus.

The dsRNA containing birnavirus, infectious pancreatic necrosis virus, possesses a virion-associated RNA-dependent RNA polymerase which acts both as primer and as polymerase during in vitro RNA synthesis (P. Dobos, 1995, Virology 208, 19-25). Using [alpha 32P]GTP, we have radiolabeled virion RNA in vitro and found that after deproteinization most of the labeled product comigrated in agarose gels with the 3-kbp viral genome, while the remainder migrated faster than the dsRNA and as a heterogeneous smear. Agarose gel electrophoresis (AGE) of denatured labeled virion RNA showed a radioactive smear ranging from approximately 100 nucleotides to up to 3000 nucleotide the size of genome length single stranded RNA. Hybridization experiments using strand-specific and end-specific obligonucleotides on Northern blots revealed that the radioactivity which migrates with the dsRNA during AGE represents small, 5' end plus RNA molecules of 100-500 nucleotides. The radioactivity in the faster migrating smear denotes incomplete dsRNAs where full-length, unlabeled minus strands are base-paired with labeled plus strands that are 3' truncated to different extents. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using end- and strand-specific obligonucleotide primers. The results indicated that 95% of incomplete dsRNA molecules consisted of full-length minus strands and 3' truncated plus strands. The implications of these findings are discussed in light of RNA replication mechanisms of dsRNA viruses belonging to other families.

Base Sequence↗
PubMed2000

Synthesis in Escherichia coli of avian reovirus core protein varsigmaA and its dsRNA-binding activity.

The genome segment S2 of p6ian reovirus (ARV) S1133 was cloned and sequenced. The entire S2 nucleotide sequence is 1325 bp long with one long open reading frame that encodes a protein of 415 amino acids, corresponding to varsigmaA, a major core protein of ARV. S2 possesses a pentanucleotide, TCATC, at the 3'-terminus of its plus strand, common to other known genome segments of ARV and to 10 genome segments of mammalian reovirus. Amino acid sequence analysis revealed that varsigmaA contains a carboxy-terminal region (one-fourth of the protein) that is formed from alpha-helices and beta-turns, and the remainder (three-fourths of the protein) is formed predominantly from beta-strands and beta-turns. Analysis of binding activity to poly(rI)-poly(rC)-agarose suggested that ARV protein A present in total virus-infected chicken embryo fibroblasts (CEF) had dsRNA-binding activity. To further characterize the binding activity, protein varsigmaA was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chelate affinity chromatography. The expressed protein evarsigmaA was further purified through a Superdex 75 HR 10/30 column after digestion of the purified fusion peptide with enterokinase. The expressed protein evarsigmaA has the same molecular weight as virion protein varsigmaA purified from ARV-infected CEF and is indistinguishable from virion protein varsigmaA by immunoblot analysis. The evarsigmaA binds cooperatively alpha (32)P-labeled dsRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reaction is blocked by homologous ARV dsRNA or heterologous infectious bursal disease virus dsRNA and poly(rI)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein evarsigmaA has dsRNA-binding activity similar to that of varsigmaA obtained from infected cells, and its binding is sequence-independent.

Amino Acid Sequence↗
PubMed1987

Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome.

Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated Ly. We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VLy-P1) and 77 kd (VLy-P2). Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz. Ly-P1 (83 kd); Ly-P2 (77 kd); Ly-P3 (74 kd) and Ly-P4 (68 kd). The in vivo viral-associated protein VLy-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product Ly-P1 and, similarly, VLy-P2 co-migrated with Ly-P2. Peptide mapping data confirm the identity of the in vivo products (VLy-P1 and VLy-P2) and their in vitro counterparts. The conclusion made is that VLy-P1 and VLy-P2 are almost identical primary translation products of the Ly genome, derived from a single or multiple species of Ly-dsRNA. RNA blot hybridizations using L1A M1 and separately, L2A M2 probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to Ly-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S. cerevisiae dsRNA killer systems.

Animals↗