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PubMed1989

Detection of killer-independent dsRNA plasmids in Ustilago maydis by a simple and rapid method of extraction of dsRNA.

A novel method for efficient and rapid isolation of dsRNA molecules was developed. The dsRNA content of Ustilago maydis was reexamined; two distinct dsRNA classes were identified. Class I includes the dsRNA segments reported earlier for U. maydis virus systems and class II includes unencapsidated dsRNA molecules that were barely detected by the conventional extraction methods despite their high titer. Segments of the class II, some of which are reported for the first time, were further characterized; all the segments are independent of the killer system and other encapsidated dsRNA molecules. These segments are cytoplasmically transmitted and, in sharp contrast with class I-encapsidated dsRNA segments, their relative copy number decreases rapidly while entering the stationary phase.

Basidiomycota↗

PubMed1989

Production of interferon-alpha induced by dsRNA in human peripheral blood mononuclear cell cultures: role of priming by dsRNA-induced interferons-gamma and -beta.

Poly(I):poly(C) (rIn.rCn), mismatched poly(I):poly(C) [rIn.r(C12U)n], and poly(I):poly(C) poly-lysine carboxymethylcellulose [poly(ICLC)] were studied for their capacity to induce interferon-alpha (IFN-alpha) in human peripheral blood mononuclear cell (MNC) cultures and in their subpopulations. In MNC, poly(I):poly(C) and mismatched poly(I):poly(C) induced IFN-alpha in a dose-dependent manner, whereas poly(ICLC) was unable to do so in concentrations that ranged from 1 to 160 micrograms/ml. In contrast, all three molecules were incapable of inducing IFN-alpha when added into either purified monocyte or lymphocyte cultures. The capacity of poly(I):poly(C) to induce IFN-alpha was reestablished only in monocyte cultures when, prior to the stimulation, the cells were exposed for at least 2 h to supernatants from poly(I):poly(C)-stimulated lymphocyte cultures. IFN-beta and IFN-gamma were found in those supernatants and the ability of each dsRNA to induce IFN-alpha correlated with its ability to induce IFN-gamma. Further studies using recombinant human IFN-gamma and IFN-beta and their specific antibodies corroborated an important role of these molecules for the induction of IFN-alpha with dsRNA. Because each IFN type is produced by different cells, our studies show that complex cell-to-cell interactions via other IFNs have to take place in peripheral blood mononuclear cells (MNC) cultures before IFN-alpha can be induced by dsRNA.

Carboxymethylcellulose Sodium↗

PubMed1992

T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae. Identification of 23 S RNA as the single-stranded form of T dsRNA.

Some strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called T. We obtained T cDNA clones by random priming of denatured T dsRNA followed by reverse transcription. Sequence data of T show that only one strand ((+)-strand) has coding capacity for a protein with 940 amino acids which spans almost the entire length of the molecule (2.9 kilobases). Within this protein we found a sequence pattern characteristic of RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. Although T has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC, M1, or W, the T-encoded protein shows a high degree of conservation with the W-encoded protein. This conservation extends beyond a region that contains the consensus sequences for RNA-dependent RNA polymerases, suggesting that both T and W are evolutionarily related. With a (+)-strand-specific probe for T we identified 23 S RNA, a new single-stranded RNA (ssRNA) species with a sedimentation coefficient of 23 S. T and 23 S RNA have the same mobility under denaturing conditions with glyoxal, suggesting that 23 S RNA is, in fact, the (+)-single-stranded RNA form of T dsRNA. 23 S RNA synthesis is induced under stress conditions such as heat shock and starvation. The relationship between T and 23 S RNA clearly resembles the one between W and its single-stranded derivative form, 20 S RNA. Thus T and W dsRNAs (and their respective single-stranded species) constitute a new RNA family in S. cerevisiae.

Amino Acid Sequence↗

PubMed1977

Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae.

Virus-like particles containing the L (P1)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [35S]methionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA genome encodes the major virus-like particle capsid polypeptide. This result adds considerable support to the hypothesis that the L-dsRNA genome acts as a helper genome to the smaller (1.6 x 10(6) dalton) M-dsRNA genome in killer strains of yeast by providing the M-dsRNA containing virus-like particles with their major coat protein.

Capsid↗

PubMed1985

The adjuvant effects of mycoviral dsRNA and polyinosinic:polycytidylic acid on the murine immune response.

By comparing, under the same experimental conditions, the effects of naturally occurring mycoviral dsRNA with those of the synthetic dsRNA, polyinosinic:polycytidylic acid, we were able to determine if the source of the dsRNA would modify its immunomodulating properties. Mycoviral dsRNA, but not the synthetic dsRNA, significantly enhanced the hemagglutinating antibody response to sRBC in C57B1/6 mice. Although both dsRNA preparations significantly increased the rate of rejection of heterologous skin grafts by recipient mice when compared to controls, mycoviral dsRNA induced higher interferon titers than the synthetic dsRNA. This study showed that mycoviral dsRNA was a more potent adjuvant than polyinosinic:polycytidylic acid for both humoral and cellular immune responses.

Adjuvants, Immunologic↗

PubMed1987

Diversity of citrus tristeza virus isolates indicated by dsRNA analysis.

One major dsRNA of molecular weight (MW) 13.3 X 10(6) and two others (MW 1.9 X 10(6) and 0.8 X 10(6] were routinely detected by polyacrylamide gel electrophoresis in extracts from sweet orange (Citrus sinensis) or citron (Citrus medica) infected with each of 66 isolates of citrus tristeza virus (CTV). Several additional dsRNA were also commonly detected, usually as weakly stained bands in reproducible positions in gels, but some were very prominent, e.g., a dsRNA of MW 1.7 X 10(6) associated with a seedling yellows isolate (sy-1). No dsRNA was detected in equivalent extracts from noninoculated sweet orange and citron. End-labeled [32P] probes were made from purified full-length viral RNA or polyacrylamide gel-purified full-length dsRNA of a nonseedling yellows (nsy-1) and a seedling yellows (sy-1) isolate of CTV. Each of the four probes was able to hybridize to all major and most minor dsRNAs of both isolates in composite polyacrylamide/agrarose gels, including the 1.7 X 10(6) dsRNA specific to the seedling yellows isolate, and could readily detect CTV nucleic acid sequences in extracts from bark of infected sweet orange plants spotted onto nitrocellulose membranes. One dsRNA (MW 0.5 X 10(6] was very prominent in some isolates and much less so, or undetectable, in other isolates and 66 isolates have been screened for the presence of this dsRNA. There was a strong correlation between inability to detect the 0.5 X 10(6) dsRNA and the designation of an isolate as neither a seedling yellows type nor a stem pitting isolate of grapefruit; these properties were typical for isolates of CTV from southern California.

Citrus↗

PubMed2026

Chitosan-dsRNA improves tissue stability and delivery for RNAi-mediated Varroa destructor control.

BACKGROUND: Varroa destructor is an ectoparasitic mite and a major threat to honey-bee colony health worldwide. RNA interference (RNAi) offers a potentially species-specific approach for mite control, but practical application is limited by double-stranded RNA (dsRNA) degradation and inefficient delivery to mites. This study evaluated coatomer protein I (COPI) complex subunits as RNAi targets and tested whether chitosan-based dsRNA formulation could improve dsRNA stability, tissue uptake, and delivery from honey-bees to mites. RESULTS: Direct microinjection of dsRNAs targeting COPB, COPD, and COPE significantly reduced target-gene expression and mite survival compared with the double-stranded green fluorescent protein (dsGFP) control, with 72-h survival rates of 8.0%, 12.7%, and 5.3%, respectively, compared with 40.7% in the control group (all log-rank P < 0.0001). Chitosan-conjugated dsRNA remained detectable for longer periods than naked dsRNA in honey-bee tissue fluids, and CNP-Cy3-dsGFP was detected in the honey-bee midgut and fat body. A qualitative fluorescence observation in V. destructor was consistent with host-to-mite dsRNA transfer. Ingestion of COP-targeted chitosan-dsRNAs reduced mite survival, whereas honey-bee survival and expression of honey-bee COP orthologs were not affected. In silico analysis detected no contiguous ≥19-nt matches between Varroa COP dsRNAs and the honey-bee transcriptome or genome. CONCLUSION: COPI subunits are promising RNAi targets in V. destructor, and chitosan formulation may improve dsRNA persistence and uptake while supporting honey-bee-mediated delivery to mites. These laboratory findings support further evaluation of chitosan-formulated dsRNA as a potentially species-selective strategy for Varroa management, while broader safety assessment and field validation remain necessary. © 2026 Society of Chemical Industry.

COPI complex↗

PubMed1988

Transcription and in vitro translation of the dsRNA virus isolated from Rhizoctonia solani.

A segmented double-stranded dsRNA virus has been isolated from virulent strains of Rhizoctonia solani. The dsRNA genome has mol. wts. of 1.45 and 1.32 X 10(6). Two full-size transcripts with mol. wts. of 0.74 and 0.66 X 10(6) (2.2 kb and 2 kb, respectively) were synthesized by the virus-associated RNA-dependent RNA polymerase and resolved by denaturing polyacrylamide gel electrophoresis. The transcripts cross-hybridized to the viral dsRNA isolated from a number of strains. The transcripts did not hybridize with the genomic DNA. An unencapsidated species of dsRNA with mol. wt. of 1.6 X 10(6) did not hybridize with the viral transcripts. No cross-hybridization between the two viral dsRNA segments was obtained. The viral-encoded proteins were studied by in vitro translation using the rabbit reticulocyte lysate system. The transcripts served as mRNA for the synthesis of the major capsid protein of 55 kD, and a number of other products. The viral coat protein was immunoprecipitated with antibodies against purified virus particles. Partial proteolysis of the major in vitro product and the authentic capsid protein using Staphylococcus aureus V8 protease produced similar peptide patterns. Denatured viral dsRNA also directed the synthesis of proteins identical to those translated from the transcripts in vitro.

Animals↗

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↗

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↗

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↗

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↗

PubMed1990

Molecular cloning and comparative sequence analyses of bluetongue virus S1 segments by selective synthesis of specific full-length DNA copies of dsRNA genes.

Using primers complementary to the conserved sequences of the 3' ends of bluetongue virus genomic dsRNA segments, full-length DNA clones of all 10 dsRNA genes from the five U.S. BTV serotypes were synthesized and amplified by a novel method (ClampR). This amounts to nearly 100,000 base pairs of dsRNA cloned as unique full-length DNA copies. This continuous one-tube procedure combined cloning of the denatured dsRNA with reverse transcriptase and the selective amplification of full-length DNA by the polymerase chain reaction. ClampR-derived clones of the genomic segment S1 of BTV-11 encoding the serogroup antigen, VP7, were sequenced and shown to be complete copy, containing a total of 1156 bp and a long open reading frame of 349 amino acids. Comparative sequence analyses of BTV-11 S1 with those of the other U.S. serotypes show that 95.2% of the nucleotides are conserved between BTV-11 and -10, while only 79.0% of the bases are identical between BTV-11 and -13. Comparison of the VP7 proteins demonstrates that 100% of the amino acids are conserved between BTV-11 and -10 and 93.7% of these residues are identical between VP7 of BTV-11 and -13. The adaptation of the polymerase chain reaction to the full-length cloning and amplification of dsRNA (ClampR) should greatly facilitate molecular studies within the Reoviridae family.

Amino Acid Sequence↗

PubMed1991

Detection of toxic viral-associated double-stranded RNA (dsRNA) in influenza-infected lung.

While many of the molecular events in viral replication are well studied, the molecular mechanisms by which viral infections trigger such constitutional symptoms as fever and 'malaise' are unknown. The hypothesis that these viral constitutional symptoms can be triggered by the toxic action of dsRNA associated with viral replication was investigated. Total lung RNA from mice acutely infected with PR8 influenza virus, but not from sham-infected mice, was shown to induce fever and altered sleep (excess slow-wave sleep, enhanced amplitudes of electroencephalographic slow waves, and reduced rapid eye movement sleep) when injected into the rabbit brain. Viral-associated dsRNA was shown to be responsible for the rabbit responses by differential nuclease digestion. Influenza viral dsRNA was directly demonstrated in the active lung RNA preparations by reverse transcriptase-polymerase chain reaction techniques. The time course of the responses paralleled those seen in the same model inoculated with nanogram quantities of the synthetic dsRNA polyriboinosinic-polyribocytidylic acid and suggested that they were mediated by induced cytokines. A model for the role of viral-associated dsRNA in eliciting both local cytotoxicity and viral constitutional symptoms is presented.

Animals↗

PubMed1979

Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs. Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.

Animals↗

PubMed1987

Growth of astrocytomas in the human tumor clonogenic assay and sensitivity to mismatched dsRNA and interferons.

Nine astrocytoma specimens were received from seven patients and processed for testing in the human tumor clonogenic assay (HTCA). Cells derived from these specimens were challenged with human natural alpha-interferon (alpha-IFN) and beta interferon (beta-IFN), recombinant beta interferon (beta ser-IFN), and mismatched double-stranded (ds) RNA (Ampligen). Six of the astrocytoma specimens formed adequate colonies for drug sensitivity testing (greater than or equal to 30 colonies/plate), and all were high-grade (III-IV) tumors. Sensitivity was defined as a greater than or equal to 50% decrease in tumor colony formation following drug exposure and was observed with alpha-IFN (2/4), beta-IFN (3/4), and mismatched dsRNA (4/5) exposure. No decrease in colony growth was observed after recombinant beta ser-IFN exposure, and in 2 of 3 cases, colony formation was stimulated. The sensitivity of 75 non-CNS solid tumors to mismatched dsRNA was compared to the high-grade astrocytomas in the HTCA. Of the 10 additional histologic tumor types studied, carcinoid and renal cell carcinomas exhibited the greatest sensitivity to mismatched dsRNA: 63% and 52%, respectively. However, in comparison, 80% of the high-grade astrocytomas were sensitive, demonstrating that these gliomas are among the most sensitive of human tumors to mismatched dsRNA in vitro. Clinical trials of interferons and mismatched dsRNA, coupled with in vitro sensitivity studies, should further define their therapeutic potential.

Astrocytoma↗

PubMed2026

Differential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutants.

Recognition of double-stranded RNA (dsRNA) triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus mutants engage distinct RNA-sensing pathways and promote differential assembly of cytoplasmic RNP granules. Infection with splicing-defective ∆E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proximity labeling proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While ∆VA-induced granules were PKR-dependent, ∆E4 mutants induced RLB-like granules independently of PKR and RNase L. In cells lacking these sensors, granule assembly during ∆E4 infection coincided with translational arrest independent of eIF2α phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.

RNA, Double-Stranded↗