Characterization of the double-stranded RNA isolated from cowpea mosaic virus-infected Vigna leaves.
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Centrifugation, Density Gradient↗
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Results for “Double Stranded RNA Viruses”
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A virus with bi-segmented double-stranded RNA genome in guinea pig intestines.
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Animals↗
Hyperpotentiation by synthetic double-stranded RNA of antibody responses to influenza virus vaccine in adjuvant 65.
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Adenine Nucleotides↗
Demonstration of double-stranded ribonucleic acid in concentrates of RNA viruses.
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Animals↗
Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission.
The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to approximately 50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.
Ascomycota↗
Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus.
The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs.
Amino Acid Sequence↗
Characterization of a herpes simplex virus sequence which binds a cellular protein as either a single-stranded or double-stranded DNA or RNA.
Earlier we reported that herpes simplex virus 1 DNA contains a sequence which binds a host protein in a sequence-specific manner as either a single-stranded or a double-stranded DNA or RNA and that this sequence is located in a transcriptional unit whose RNA traverses the origin of viral DNA replication (OriSRNA) (R.J. Roller, L. McCormick, and B. Roizman, Proc. Natl. Acad. Sci. USA 86:6518-6522, 1989). The protein reacts with both DNA and RNA in band-shift assays and protects the single-stranded RNA sequence from digestion by RNase. We report that the minimal cognate sequence required for these interactions consisted of [N(GTGGGTGGG)2(N less than or equal to 10)]. The ninemer repeat sequence was located at nucleotides -1 to -18 relative to the transcription initiation of the major species of OriSRNA. The activity of the cognate sequence required at least three guanines between thymines and tolerates the insertion of additional thymines, but it was inactivated by the insertion of adenines or by the substitution of some of the guanines with cytosines in one repeat. Replacement of the 10 3' nucleotides has no effect on binding activity, whereas deletion of these sequences abolished it. Among the related sequences with no demonstrable binding activity were some telomeric sequences which interact with known cognate proteins. The electrophoretic mobility of the herpes simplex virus cognate sequences in nondenaturing gels suggests that they may be able to form higher-order structures, but the conditions under which they were formed were different from the optimal conditions for binding the protein. UV light cross-linking studies of labeled RNA-protein complexes following digestion with RNases indicated that the electrophoretic mobility of the protective activity corresponded to that of a protein with an apparent molecular weight of 100,000.
Base Sequence↗
Viral double-stranded RNA, cytokines, and the flu.
The symptoms of the flu, such as fever, drowsiness, and malaise, are the sole means by which this common clinical syndrome is defined. The syndrome is usually the first clinical manifestation of both acute bacterial and viral infections. In the case of acute bacterial infections, several proinflammatory cytokines induced by bacterial products have been implicated as the causative agents of the flu syndrome. Viruses induce similar cytokines to bacteria, plus substantial amounts of interferon-alpha (IFN-alpha), although the direct association of these cytokines with the viral flu syndrome is less clear. Furthermore, the viral inducer(s) of cytokines has not been defined. The best candidate cytokine inducer associated with a majority of viral infections is virus-associated double-stranded RNA (dsRNA). This review examines the essential physical properties of toxic dsRNA, the cytokines induced by it, its viral and cellular sources, evidence for its presence in infected cells, its quantities in normal and infected cells, its cytotoxic mechanisms, and its cell-penetration properties. Toxic effects of viruses and dsRNA are compared. Energetics and extraction artifact issues are also discussed. Whereas most research on dsRNA toxicity has employed synthetic dsRNA, studies with virus-associated dsRNA are featured when available. Finally, a model for how viral dsRNA might initiate systemic disease is presented.
Animals↗
Sequence analyses and structural predictions of double-stranded RNA segment S1 and VP7 from United States prototype bluetongue virus serotypes 13 and 10.
The nucleotide sequence of segment S1 and the deduced amino acid sequence of VP7 from bluetongue virus (BTV) serotype 13 was determined. Sequences were obtained by use of standard dideoxy DNA sequencing and by direct sequencing of genomic double-stranded RNA (dsRNA). The dsRNA was sequenced with a new dideoxy protocol that produces 300 to 350 bases per set of reactions. Segment S1 is 1156 bp long and contains one long open reading frame capable of coding for 349 amino acids. The protein, VP7, is rather hydrophobic, and has a calculated molecular weight of 38,619 and a net charge of +1.5 at pH 7.0. Segment S1 of BTV-13 has 79.6% of its nucleotides conserved when compared with segment S1 of BTV-10. While most of these differences occur at the third codon position of the open reading frame, the differences between the 89-base-long, 3' noncoding regions occur predominantly in pockets at positions 1092-1098, 1112-1114, and 1125-1129. Potential stem-loop structures encompassing the stop codon of the open reading frame are proposed for both serotypes. Comparisons of VP7 from BTV-13 and BTV-10 indicate that 93.7% of the amino acid residues are conserved, including a single lysine at position 255. Secondary structure predictions infer an eight-stranded beta-barrel structure between residues 150 and 250. This putative beta-barrel may serve as a target for the development of drugs to combat bluetongue disease. Comparable structures detected in the core proteins of single-stranded RNA viruses from both plants and animals suggest that these viruses and BTV had a common origin.
Amino Acid Sequence↗
Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus.
Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.
Animals↗
Molecular and genetic comparisons of two serotypes of epizootic hemorrhagic disease of deer virus.
The virus-specific double-stranded genome RNA of 2 serotypes of epizootic hemorrhagic disease of deer virus (EHDV) was evaluated by use of coelectrophoresis in polyacrylamide and agarose gel systems. The molecular weights of virion RNA segments were 0.32 to 2.57 X 10(6) for EHDV-1 and 0.33 to 2.54 X 10(6) for EHDV-2. Seven of 10 double-stranded RNA segments of the 2 serotypes had different electrophoretic mobilities in the polyacrylamide-gel electrophoresis system. Although the individual RNA segments of each serotype contained unique RNA sequences determined on the basis of 2-dimensional polyacrylamide-gel electrophoresis analysis of oligonucleotides, the corresponding segments of the 2 serotypes were found to be comparable and at least 1 pair of RNA segment was almost identical. Virus-specific polypeptides for the 2 serotypes were compared by use of gel electrophoresis. Eleven polypeptides were detected for EHDV-1 and 10 for EHDV-2. Six corresponding polypeptides of these 2 serotypes had different electrophoretic mobilities, indicating that these corresponding polypeptides differ in their molecular weights. A genetic relationship was not determined between the 2 EHDV serogroups and the blue-tongue serogroup viruses, using oligonucleotides mapping.
Animals↗
Interferon induction by viruses. IV. Sindbis virus: early passage defective-interfering particles induce interferon.
We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.
Animals↗
Dual utilization of an acceptor/donor splice site governs the alternative splicing of the IRF-3 gene.
Interferon regulatory factors constitute a family of transcriptional activators and repressors involved in a large number of vital cellular processes. Interferon regulatory factor-3 (IRF-3) has been implicated in virus and double-stranded RNA mediated induction of IFNbeta and RANTES, in DNA damage signaling, and in virus-induced apoptosis. With its critical role in these pathways, the activity of IRF-3 is tightly regulated in myriad ways. Here we describe novel regulation of IRF-3 at the level of RNA splicing. We show that an unprecedented dual utilization of a splice acceptor/donor site within the IRF-3 mRNA governs the production of two alternative splice isoforms.
Alternative Splicing↗
A new double-stranded RNA mycovirus from Botrytis cinerea.
A simple double-stranded RNA mycovirus was detected in a wild-type Botrytis cinerea 55k strain. The virus was located in the fungus cytoplasm as free particles of approximately 28 nm in diameter. The mycovirus possesses a single double-stranded genome segment of 1.8 kilobase pairs (kbp) encapsidated within an isometric protein coat whose main structural component is a polypeptide of 68 kDa. Cells infected with this virus showed an important degree of cellular degeneration.
Botrytis↗
Sindbis virus RNA replication. II. Strand composition and metabolic fate of the multi-stranded RNA species.
Double-stranded RNA from SB virus-infected cells was denatured and analysed on agarose-methylmercuric hydroxide gels. Equimolar amounts of three single-stranded species with mol. wt. of 4 x 10(6), 2.5 x 10(6) and 1.8 x 10(6) were found. Pulse and chase experiments in infected cells established a precursor-product relationship between the multi-stranded and single-stranded virus RNA species. The present results support the model in which the 49S and 26S species of virus RNA are synthesized in infected cells from two distinct replicating structures.
Kinetics↗
In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization.
A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.
Animals↗
Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA.
RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.
Animals↗
Defective Interference in the Killer System of Saccharomyces cerevisiae.
The K(1) killer virus (or plasmid) of Saccharomyces cerevisiae is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics 74:571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics 84:27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus (MAK genes) were needed by a suppressive mutant. No effect of ski mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.
Journal Article↗