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At least 973 records · Page 54Linked to original sources
PubMed2003

Two unrelated double-stranded RNA molecule patterns in Gremmeniella abietina type A code for putative viruses of the families Totiviridae and Partitiviridae.

Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133 bp and contained two open reading frames (ORFs). The 5'-proximal ORF coded for a putative coat protein (CP) and the 3'-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae ( Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181 bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.

Base Sequence↗
PubMed2005

Molecular characterization of a partitivirus from the plant pathogenic ascomycete Rosellinia necatrix.

The W8 isolate of the phytopathogenic fungus, Rosellinia necatrix that causes white root rot, contained three segments of double-stranded (ds) RNA, namely L1, L2 and M. Purified viral particles of about 25 nm in diameter contained an RNA segment with almost the same mobility as M-dsRNA, but the band was sensitive to S1 nuclease. Molecular analysis revealed that M-dsRNA consisted of two (RNA 1 and RNA 2) similarly sized species of 2299 and 2279 bp excluding an interrupted poly (A or U) tail of 16-51 bp. The predicted largest open reading frame in RNA 1 and RNA 2 was similar to those of RNA dependent RNA polymerase (RdRp) and coat protein (CP), respectively, encoded by the family Partitiviridae. The non-coding regions (NCR) of the two segments were similar (approximately 70% base identity) at the 5' end, but different at the 3' end. The NCR at the 3' end contained adenosine-uracil rich elements (AREs) in both segments. Northern analyses revealed RNA 1 and RNA 2 in mycelial and viral particle fractions. We coined the name Rosellinia necatrix partitivirus 1-W8 (RnPV1-W8) for M-dsRNA based on viral particle morphology and sequence information.

Amino Acid Sequence↗
PubMed2005

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yidianhong [corrected].

Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yidianhong [corrected] with yellowing at the leaf edge in China. Purified viral particles of 28-30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5' untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3' UTR, with an identical terminal motif (5'-AAAAUAAAACC-3'). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.

3' Untranslated Regions↗
PubMed2001

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

Citrus↗
PubMed2026

Endosymbiotic theory of aging revisited: Age-related leakage of mitochondrial dsDNA/RNA stimulates cytosolic nucleic acid sensors which remodel the immune network and promote the aging process.

About 1.5-2 billion years ago, an endosymbiosis between aerobic α-proteobacteria and anaerobic archaeal cells generated mitochondria, i.e., organelles capable of producing oxidative energy. The bacterial genome was fundamentally reduced and a circular mitochondrial genome evolved containing mainly the genes coding for the subunits of the electron transport chain. Before the symbiotic event, there existed a virus-host co-evolution which involved the development of sensors for detecting dangerous viral DNA/RNA molecules. Endosymbiosis supplied eukaryotic cells not only with an oxidative powerhouse to allow the evolution of more complex multicellular organisms but it also meant that cells now housed an organelle which was able to generate reactive oxygen species (ROS) and to leak mitochondrial DNA (mtDNA) and double-stranded RNA (dsRNA) into the cytoplasm. There is now abundant evidence that during aging and age-related diseases mitochondria are prone to release both mtDNA and dsRNA. In the cytoplasm, mtDNA/dsRNA molecules activate a number of cytosolic nucleic acid sensors leading to the secretion of type-1 interferons (IFN) and many other cytokines which promote an age-related proinflammatory state. Currently, it is known that mtDNA can activate the cGAS-STING pathway, AIM2 inflammasomes, IFI16 receptors, and ZBP1 sensors and in addition mitochondrial dsRNA stimulates RIG-1/MDA5 signaling. Interestingly, there is abundant evidence that all these receptors are drivers of cellular senescence and inflammaging. For decades, there has been mounting evidence that mitochondria have a crucial role in the aging process. We will examine this question from the perspective of evolution and propose that mitochondrial evolution created an endogenic source for the leakage of dangerous mtDNA/dsRNA which subsequently stimulated cytosolic DNA/RNA sensors, an evolutionarily conserved viral defence mechanism. It seems that these two evolutionary events provided not only the basis for the inevitable process of aging but also ensuring the death of parental organisms.

Aging↗
PubMed2006

Stable RNA interference in Spodoptera frugiperda cells by a DNA vector-based method.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.

Animals↗
PubMed2005

Evidence implying only unprimed RdRP activity during transitive gene silencing in plants.

RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3'-5' direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3' end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a beta-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5' fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3'-untranslated region. Conversely, silencing induced by a 3' fragment spread only for a limited distance in the 3'-5' direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3' end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.

3' Untranslated Regions↗
PubMed1990

Adapting the polymerase chain reaction to a double-stranded RNA genome.

We have adapted the polymerase chain reaction (PCR) to a double-stranded RNA (dsRNA) target without possessing unambiguous sequence information. Infectious bursal disease virus of chickens, a member of the binavirus group, has a dsRNA genome which is resistant to denaturation and subsequent enzyme modification. The only published sequence information was for a strain of virus unavailable to us. We have used a quick primer binding assay to select appropriate primers and have combined a simple denaturation method with reverse transcription and subsequent polymerization using the cDNA template to yield amplified product easily detectable by ethidium bromide staining. By varying the times of denaturation, annealing, and polymerization and by reducing the total number of amplification cycles, artifacts have been eliminated when using purified genome as the template. This allowed us to obtain partial sequence information for one viral strain. We have enhanced the utility of our method by optimizing a rapid cell lysis and capsid digestion protocol such that no purification steps are required from initial tissue handling through final PCR product. Total time for all procedures involved no more than 6 h. This technique should be applicable to all other members of the Birnaviradae family and to any other species of dsRNA.

Animals↗
PubMed1984

The capsid polypeptides of the yeast viruses.

The yeast virus is a double-stranded RNA virus with a large genomic dsRNA and one major viral capsid polypeptide. Most strains of yeast have a major and a minor species of the large genomic dsRNA present. The major species has previously been shown to encode a capsid polypeptide with a Mr of about 88,000. We show that the minor species also encodes its capsid polypeptide with a Mr of about 80,000. Unlike all the dsRNA viruses of procaryotes and higher eucaryotes, the yeast virus appears to have only one major polypeptide in its virions. There are some 60 molecules of this capsid polypeptide per particle, consistent with a simple icosohedron of T=1.

Capsid↗
PubMed1984

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases.

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.

Blood Proteins↗
PubMed1985

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.

Animals↗
PubMed1990

Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels.

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.

Autoradiography↗
PubMed1986

A mechanism for the control of protein synthesis by adenovirus VA RNAI.

In the absence of VA RNAI, protein synthesis in adenovirus-infected HeLa cells fails because of defective initiation. Earlier work showed that the defect results from phosphorylation of the initiation factor elF-2 on its alpha subunit. We have identified the protein kinase responsible as the dsRNA-activated inhibitor of protein synthesis (DAI). DAI is present in uninfected state. It is activated in cells infected with the adenovirus mutant Ad5 dl331, which produces no VA RNAI, but not in cells infected with wild-type virus. Activation occurs during the late phase of infection with the mutant virus, and the activator appears to be dsRNA produced by symmetrical transcription of the viral genome. VA RNAI antagonizes the activation of DAI by dsRNA, but it cannot inhibit the activity of DAI once activated. We propose a mechanism for VA RNAI action based on its partially double-stranded nature.

Adenoviruses, Human↗
PubMed1990

Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function.

Adenovirus VA RNAL maintains protein synthesis by preventing activation of the double-stranded RNA (dsRNA)-dependent protein kinase DAI. There appears to be a single binding site for dsRNA on DAI, and this site is blocked by VA RNAl. VA RNAl binds to purified DAI and can be cross-linked to the enzyme by UV irradiation. To determine the relationship between DAI binding and VA RNAl structure and function, we examined the binding abilities of wild-type and mutant VA RNAs. In several cases, the ability to bind DAI efficiently in vitro did not correlate with function in vivo. Secondary structure analysis suggested that efficient binding requires an apical stem-loop structure, whereas inhibition of DAI activation requires the central domain of the VA RNA molecule. We propose that the duplex stem permits VA RNA to interact with the dsRNA binding site on DAI and inhibits activation by juxtaposing the central domain of the RNA with the enzyme's active site.

Adenoviruses, Human↗
PubMed1990

Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection.

The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.

Electrochemistry↗
PubMed1982

Cloning of cDNA to a yeast viral double-stranded RNA and comparison of three viral RNAs.

We have constructed recombinant DNA clones containing small complementary DNA (cDNA) sequences homologous to portions of a 4.8-kb yeast viral double-stranded RNA (dsRNA) (L1) that codes for the viral capsid polypeptide. Neither the viral dsRNA nor its in vitro transcript is polyadenylated; hence the cDNAs were synthesized by reverse transcriptase on the in vitro mRNA transcript made by the viral transcriptase, using sheared salmon sperm DNA as a random primer. This is the first reported cloning of cDNA homologous to a viral double-stranded RNA. This method should be of general utility for dsRNA viruses, since all have a capsid-associated transcriptase activity. The lengths of the overlapping cDNA inserts varied from 100 to 800 bp. About 40% of them mapped to the 5' end of the in vitro transcript, and these have been ordered. At least 1485 bp of this end of L1 is represented in the cloned cDNAs characterized. Using the cloned cDNAs as probes, we have shown that the L dsRNAs of two viral subtypes are similar at the transcription initiation site and dissimilar elsewhere.

Cloning, Molecular↗
PubMed1982

Electrophoretic comparison of the genomes of North American bluetongue viruses, one Australian bluetongue virus, and three other related orbiviruses.

The genomes of U.S. bluetongue viruses, an Australian bluetongue virus, and three other related orbiviruses were analyzed by polyacrylamide gel electrophoresis. The genomes were comprised of ten segments of double-stranded (ds) RNA. Estimates of the molecular weights of the dsRNA segments revealed that the U.S. bluetongue serotypes were remarkably similar. Although the dsRNA profiles of the viruses exhibited common segments, each virus had a distinct dsRNA profile. The usefulness of the genome analysis as a diagnostic tool for identification and for epidemiologic studies is discussed.

Arboviruses↗
PubMed2004

Langerhans cells exhibit low responsiveness to double-stranded RNA.

Double-stranded RNA (dsRNA) is a viral product recognized by Toll-like receptor 3 (TLR3), and it is a potent activator of dendritic cells (DC). We compared Langerhans cells (LC) and splenic CD11c(+) DC and investigated the responsiveness to dsRNA. We prepared highly purified LC (> 95%) using the panning method. TLR3 mRNA was expressed in LC, splenic DC, and keratinocytes (KC). The expression of IFN-beta mRNA was enhanced in LC and splenic DC by Poly(I:C) stimulation. However, cytokine/chemokine production in response to Poly(I:C) by LC was much lower than that by splenic DC. In addition, Poly(I:C) induced further maturation in splenic DC, but not in LC. Finally, we found that the mouse KC cell line, PAM212, produced a great amount of IL-1alpha by Poly(I:C) stimulation, and that IL-1alpha promoted the maturation of LC. These data altogether indicate that LC exhibit low responsiveness to dsRNA. It is possible that KC may primarily trigger anti-viral immune responses in the skin via cytokine production such as IL-1alpha.

Animals↗