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PubMed1979

Evidence for secondary structure in poliovirus virion RNA demonstrated by antibodies against double-stranded RNA.

Poliovirus particle RNA has been considered to have little secondary structure. Specific binding of poliovirion RNA by antibodies against double-stranded RNA (dsRNA) has been confirmed and further characterized by a radioimmunoassay using Staphylococcus aureus protein A to precipitate the nucleic acid-antibody complex. Competitive binding studies between virion single-stranded RNA (ssRNA) and poly(I).poly(C) demonstrated that the dsRNA effectively inhibited binding of radiolabelled poliovirion RNA by the anti-dsRNA antibodies but the virion RNA was a poor competitor of radiolabelled ds RNA. This indicates that both RNAs reacted with the same species of antibodies in the sera, but avidity of the antibodies for dsRNA was greater than for the poliovirion RNA.

Animals↗
PubMed1980

A comparative study on the u.v. resistance of double-stranded and single-stranded encephalomyocarditis virus RNAs: evaluation of the possible contribution of host-mediated repair.

To reveal previously suggested host-mediated repair of u.v.-induced lesions in dsRNA of encephalomyocarditis (EMC) virus, two sets of experiments have been carried out: (i) samples of dsRNA of EMC virus were irradiated with different doses of u.v. light and their infectivity was assayed in krebs II cells, before and after conversion of dsRNA into a ss form: (ii) samples of ssRNA of EMS virus were similarly irradiated and their infectivity was assayed before and after conversion of ssRNA into a ds form. No evidence for a significant host-mediated repair of dsRNA in this virus-cell system has been obtained.

Animals↗
PubMed1998

Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the '447' cytoplasmic male sterility trait in Vicia faba.

The entire nucleotide sequence of the double-stranded (ds) RNA associated with the unconventional '447' cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17,635 nt long, while the positive-strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14,900 nt, with the smaller RNA being located on the 5' side. The entire positive-strand could encode a single in-frame ORF starting at the first AUG at position 42-44 and ending with a TGA at 17,517-17,519. This long potential polypeptide with a predicted molecular mass of 654,109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by self-cleavage being the determinant of the CMS trait.

Amino Acid Sequence↗
PubMed2005

Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Adenosine Triphosphatases↗
PubMed2004

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus.

Molecular cloning and complete nucleotide sequencing of Penicillium chrysogenum virus (PcV) dsRNAs indicated that PcV virions contained four dsRNA segments with sizes of 3562, 3200, 2976 and 2902 bp. Each dsRNA segment had unique sequences and contained a single large open reading frame (ORF). In vitro translation of transcripts derived from full-length cDNA clones of PcV dsRNAs yielded single products of sizes similar to those predicted from the deduced amino acid sequences of the individual ORFs. Sequence similarity searches revealed that dsRNA1 encodes a putative RNA-dependent RNA polymerase. In this study, it was determined that dsRNA2 encodes the major capsid protein and that p4, encoded by dsRNA4, is virion-associated as a minor component. All four dsRNAs of PcV, like the genomic segments of viruses with multipartite genomes, were found to have extended regions of highly conserved terminal sequences at both ends. In addition to the strictly conserved 5'-terminal 10 nt, a second region consisting of reiteration of the sequence CAA was found immediately upstream of the AUG initiator codon. These (CAA)(n) repeats are reminiscent of the translational enhancer elements of tobamoviruses. The 3'-terminal 14 nt were also strictly conserved. As PcV and related viruses with four dsRNA segments (genus Chrysovirus) have not been previously characterized at the molecular level, they were provisionally classified in the family Partitiviridae, comprising viruses with bipartite genomes. This study represents the first report on molecular characterization of a chrysovirus and the results suggest the creation of a new family of mycoviruses with multipartite dsRNA genomes to accommodate PcV and related viruses.

Amino Acid Sequence↗
PubMed2005

A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene.

A new dsRNA was isolated from a Phytophthora isolate from Douglas fir. Sequence analysis showed the dsRNA to consist of 13 883 bp and to contain a single open reading frame with the potential to encode a polyprotein of 4548 aa. This polyprotein contained amino acid sequence motifs characteristic of virus RNA-dependent RNA polymerases (RdRps) in its C-terminal region and motifs characteristic of RNA helicases in its N-terminal region. These sequence motifs were related to corresponding motifs in plant viruses in the genus Endornavirus. In phylogenetic trees constructed from the RdRp and helicase motifs of a range of ssRNA and dsRNA viruses, the Phytophthora RdRp and helicase sequences clustered with those of the plant endornaviruses with good bootstrap support. The properties of the Phytophthora dsRNA are consistent with its being classified as the first non-plant member of the genus Endornavirus, for which we propose the name phytophthora endornavirus 1 (PEV1). A region between the RdRp and helicase domains of the PEV1 protein had significant amino acid sequence similarity to UDP glycosyltransferases (UGTs). Two sequence motifs were identified, one characteristic of all UGTs and the other characteristic of sterol UGTs. The PEV1 UGT would be the first for an RNA virus, although ecdysteroid UGT genes have been found in many baculoviruses. The PEV1 UGT was only distantly related to baculovirus ecdysteroid UGTs, which belong to a family distinct from the sterol UGTs.

Amino Acid Motifs↗
PubMed1991

Inhibitor of interferon-induced double-stranded RNA-dependent protein kinase and its relevance to alteration of cellular protein kinase activity level in response to external stimuli.

In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.

Antiviral Agents↗
PubMed2002

Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication.

RNA interference (RNAi) has been reported to be post-transcriptional gene silencing (PTGS) by approximately 500 nucleotide-(nt)-long double-stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV-1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long-term non-progressors (LTNPs) using cotransfection of the target gene-expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV-1 (SF2) LTR in persistently HIV-1-infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full-length nef dsRNA (744 nt), suggesting that approximately 500 nt-long nef dsRNA could interfere with the HIV-1 transcription.

Gene Products, nef↗
PubMed1987

The double-stranded RNA-dependent protein kinase is also activated by heparin.

The double-stranded(ds)-RNA dependent protein kinase from human cells is a Mr 68,000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin III, heparin-binding protein. However, when antithrombin III is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-azido-[alpha-32P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.

Anions↗
PubMed1995

Double-stranded RNA mycoviruses in mycelium of Pleurotus ostreatus.

Mycelium of Pleurotus ostreatus var. florida with a decreased growth rate contained seven double-stranded RNA segments and isometrical virus particles with diameters of 24 and 30 nm. Mycelium with a normal growth rate lacked dsRNA. Protoclones from virus-containing mycelium contained one to seven of these dsRNA segments in varying concentrations. The exact correlation between slow growth and the presence of dsRNA molecules could not be established. Infection of virus-free protoplasts with PEG-precipitated virus particles resulted in mycelium that stably maintained the 2.4 kbp dsRNA.

Electrophoresis, Agar Gel↗
PubMed2005

Identification of the heparin-binding domains of the interferon-induced protein kinase, PKR.

PKR is an interferon-induced serine-threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR's kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double-stranded (ds) RNA serves as PKR's activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP-binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the alpha-subunit of the protein synthesis initiation factor 2 (eIF2alpha) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA-induced activation of PKR, heparin-dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin-induced PKR activation, we have mapped the heparin-binding domains of PKR. Our results indicate that PKR has two heparin-binding domains that are nonoverlapping with its dsRNA-binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin-binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.

Amino Acid Sequence↗
PubMed2021

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Ascomycota↗
PubMed1992

Temperature and structural effects on transfer of double-stranded RNA among isolates of the chestnut blight fungus (Cryphonectria parasitica).

Cryphonectria parasitica is a unique fungus which can serve as a model for understanding transfer of genes between eukaryotic microorganisms. We studied transfer of double-stranded RNA (dsRNA) between compatible and incompatible strains of C. parasitica to determine whether hyphal types or temperature could restrict that exchange. Hyphal connections between incompatible strains occurred at about 30% of the frequency of connections between compatible strains and differed morphologically. Gel electrophoresis and in situ hybridization confirmed that dsRNA was transferred through substrate hyphae but not through aerial hyphae. Freezing temperatures resulted in the loss of dsRNA from the new mycelium of the donor colony and stimulated the production of virulent pycnidiospores. These temperature and structural restrictions may help to explain the lack of spread of the dsRNA despite its presence in the field.

Ascomycota↗
PubMed2006

Suppression of proinflammatory signal transduction and gene expression by the dual nucleic acid binding domains of the vaccinia virus E3L proteins.

Cells have evolved elaborate mechanisms to counteract the onslaught of viral infections. To activate these defenses, the viral threat must be recognized. Danger signals, or pathogen-associated molecular patterns, that are induced by pathogens include double-stranded RNA (dsRNA), viral single-stranded RNA, glycolipids, and CpG DNA. Understanding the signal transduction pathways activated and host gene expression induced by these danger signals is vital to understanding virus-host interactions. The vaccinia virus E3L protein is involved in blocking the host antiviral response and increasing pathogenesis, functions that map to separate C-terminal dsRNA- and N-terminal Z-DNA-binding domains. Viruses containing mutations in these domains allow modeling of the role of dsRNA and Z-form nucleic acid in the host response to virus infection. Deletions in the Z-DNA- or dsRNA-binding domains led to activation of signal transduction cascades and up-regulation of host gene expression, with many genes involved in the inflammatory response. These data suggest that poxviruses actively inhibit cellular recognition of viral danger signals and the subsequent cellular response to the viral threat.

DNA-Binding Proteins↗
PubMed2006

RNA- and virus-independent inhibition of antiviral signaling by RNA helicase LGP2.

Antiviral innate immune responses can be triggered by accumulation of intracellular nucleic acids resulting from virus infections. Double-stranded RNA (dsRNA) can be detected by the cytoplasmic RNA helicase proteins RIG-I and MDA5, two proteins that share sequence similarities within a caspase recruitment domain (CARD) and a DExD/H box RNA helicase domain. These proteins are considered dsRNA sensors and are thought to transmit the signal to the mitochondrial adapter, IPS-1 (also known as MAVS, VISA, or CARDIF) via CARD interactions. IPS-1 coordinates the activity of protein kinases that activate transcription factors needed to induce beta interferon (IFN-beta) gene transcription. Another helicase protein, LGP2, lacks the CARD region and does not activate IFN-beta gene expression. LGP2 mRNA is induced by interferon, dsRNA treatments, or Sendai virus infection and acts as a feedback inhibitor for antiviral signaling. Results indicate that LGP2 can inhibit antiviral signaling independently of dsRNA or virus infection intermediates by engaging in a protein complex with IPS-1. Experiments suggest that LGP2 can compete with the kinase IKKi (also known as IKKepsilon) for a common interaction site on IPS-1. These results provide the first demonstration of protein interaction as an element of negative-feedback regulation of intracellular antiviral signaling by LGP2.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection.

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.

Adenosine Deaminase↗
PubMed1972

Mechanism of Mengo virus-induced cell injury in L cells: use of inhibitors of protein synthesis to dissociate virus-specific events.

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.

Amino Acids↗
PubMed1974

Shythesis of reovirus oligo adenylic acid in vivo and in vitro.

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 mug of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The (3)H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of approximately 45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins.

Adenosine Monophosphate↗