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At least 955 records · Page 53Linked to original sources
PubMed1998

Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A.

In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.

Animals↗
PubMed1998

Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome.

The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2 (Ld), of the ascomycete fungus Ophiostoma novo-ulmi has been determined. One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins of 178 and 182 amino acids, respectively. The large ORF contains 12 UGA codons which code for tryptophan in ascomycete mitochondria and has a codon bias typical of mitochondrial genes, consistent with the localization of Ld dsRNAs within the mitochondria. The amino acid sequence contains motifs characteristic of RNA-dependent RNA polymerases (RdRps). This putative RdRp was shown to be related to putative RdRps of mitochondrial dsRNAs of another ascomycete and a basidiomycete fungus and also to a putative RdRp encoded by the mitochondrial genome of Arabidopsis thaliana. In multiple sequence alignments, the fungal mitochondrial dsRNA-encoded RdRp-like proteins formed a cluster, ancestrally related to the RdRps of the yeast 20S and 23S RNA replicons and of the positive-stranded RNA bacteriophages of the Leviviridae family, but distinct from RdRps of other families and genera of fungal RNA viruses and related plant and animal RNA viruses. Northern blot analysis with RNA 3a strand-specific probes indicated that nucleic acid extracts of Ld contain more single-stranded (positive-stranded) RNA than dsRNA, consistent with an evolutionary relationship between RNA 3a and positive-stranded RNA phages.

Amino Acid Sequence↗
PubMed1999

Nucleoside triphosphatase and RNA helicase activities associated with GB virus B nonstructural protein 3.

GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Flaviviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase. The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease. In this report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity. This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities. Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) "AXXXXGK(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Kinetic parameters (K(m) and k(cat)) for hydrolysis of various NTPs or dNTPs were obtained. The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3' unpaired regions (RNAs with 5' unpaired regions only or with blunt ends are not suitable substrates for this enzyme). This indicates that GBV-B NS3 RNA helicase unwinds dsRNA in the 3' to 5' direction. Direct interaction of the GBV-B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay. Finally, a homology model of GBV-B NS3 RNA helicase domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed.

Acid Anhydride Hydrolases↗
PubMed2001

Identification of sequences in rotavirus mRNAs important for minus strand synthesis using antisense oligonucleotides.

The core of the rotavirion consists of three proteins, including the viral RNA polymerase, and 11 segments of double-stranded (ds)RNA. The RNA polymerase of disrupted (open) cores is able to catalyze the synthesis of dsRNA from exogenous viral mRNAs in vitro. In this study, we have identified sequences in exogenous viral mRNAs important for RNA replication using antisense oligonucleotides. The results showed that oligonucleotides complementary to the highly conserved 3'-terminal sequence of rotavirus mRNAs prevented all but basal levels of dsRNA synthesis. Notably, we observed that the addition of oligonucleotides which were complementary to nonconserved sequences present either at the 5'- or 3'-end of a viral mRNA effectively inhibited its replication without interfering with the replication of other viral mRNAs present in the same replication assay. Thus, the nonconserved sequences in rotavirus mRNAs contain gene-specific information that promotes RNA replication. The fact that antisense oligonucleotides inhibited dsRNA synthesis indicates that the strandedness (single- versus double-stranded) and secondary structure of the viral mRNA template are factors that affect the efficiency of minus strand synthesis.

3' Untranslated Regions↗
PubMed2002

Interferon regulatory factor-1, interferon-beta, and reovirus-induced myocarditis.

Viral myocarditis is an important human disease, and reovirus-induced myocarditis in mice provides an excellent model to study direct viral damage to the heart. Previously, we showed that reovirus induction of and sensitivity to interferon-beta (IFN-beta) is an important determinant of viral pathogenicity in the heart and that the transcription factor interferon regulatory factor-3 (IRF-3) is required for reovirus induction of IFN-beta in primary cardiac myocyte cultures. Given several lines of evidence suggesting a possible distinctive environment for IRFs in the heart, we have now focused on IRF-1. Previous studies demonstrated that viruses, double-stranded-RNA (dsRNA), and IFN-alpha/beta can each induce IRF-1 and that IRF-1 plays a role in dsRNA, but perhaps not viral, induction of IFN-alpha/beta. Importantly, none of these studies used a virus with a dsRNA genome (such as reovirus), none of them used a highly differentiated nonlymphoid cell type, and none of them addressed whether viral induction of IRF-1 is direct or is mediated through viral induction of IFN-beta. Indeed, as recently as this year it has been assumed that viral induction of IRF-1 is direct. Here, we found that reovirus induced IRF-1 in primary cardiac myocyte cultures, but that IRF-1 was not required for reovirus induction of IFN-beta. Surprisingly, we found that reovirus failed to induce IRF-1 in the absence of the IFN-alpha/beta response. This provides the first evidence that viruses may not induce IRF-1 directly. Finally, nonmyocarditic reovirus strains induced more cardiac lesions in mice deficient for IRF-1 than they did in wildtype mice, directly demonstrating a protective role for IRF-1. Together, the results indicate that while IRF-1 is downstream of the IFN-beta response, it plays an important protective role against viral myocarditis.

Animals↗
PubMed2002

The V proteins of simian virus 5 and other paramyxoviruses inhibit induction of interferon-beta.

In this article we show that the paramyxovirus SV5 is a poor inducer of interferon-beta (IFN-beta). This inefficient induction is a consequence of the expression of an intact viral V protein. In the absence of the viral V protein cysteine-rich C-terminal domain, IFN-beta mRNA is strongly induced and the transcription factors NF-kappaB and IRF-3 are activated significantly. The V protein can work in isolation from SV5 to block intracellular dsRNA signaling. The mechanism of block to dsRNA signaling is distinct from that previously observed for blocking IFN signaling in that proteolysis of candidate factors cannot be detected, and furthermore, the respective blocks require distinct protein domains. Blocking of the induction of IFN-beta by dsRNA requires the C-terminal cysteine-rich domain, a feature that is highly conserved among paramyxoviruses. We demonstrate that the V proteins from other paramyxoviruses have equivalent functions and speculate that limiting the yield of IFN-beta during infection may be a general property of paramyxoviruses.

Animals↗
PubMed2005

Genetic analysis of innate immunity: identification and function of the TIR adapter proteins.

The innate immune system senses pathogens largely through signals initiated by a collection of phylogenetically related proteins known as "Toll-like receptors" (TLRs), of which ten representatives are encoded in the human genome. The sensing role of the TLRs first came to light when one member of this family, TLR4, was shown to serve the detection of endotoxin (lipopolysaccharide; LPS) in mice. This discovery was based upon positional cloning of a spontaneous mutation affecting a locus known as Lps. The recognition specificities of other TLRs have since been established by reverse genetic methods. The understanding of the biochemical circuitry that maintains the innate capacity for immune recognition and response has loomed as the next hurdle in the field. A total of five adapter proteins with cytoplasmic domain homology to the TLRs are known to exist in mammals. These proteins are not entirely promiscuous in their interaction with TLRs, but rather, show preferential association with individual family members, giving a particular character to the signals that distinct micro-organisms initiate. The adaptive immune response is dependent upon upregulation of costimulatory molecules (UCM) such as CD80 and CD86. Very recently, it has been shown that this upregulation is dependent upon an adapter encoded by a locus known as Lps2, known as Trif or Ticam-1, and upon type I interferon receptor signaling. LPS and dsRNA both signal via Trif, but dsRNA has an accessory pathway for UCM, that is independent of both Trif/Ticam-1 and the known dsRNA receptor, TLR3. Other key innate immunity genes have also been disclosed by germline mutagenesis, and are discussed in the present review.

Adaptor Proteins, Signal Transducing↗
PubMed1980

Double-stranded RNA.

High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplornaviruses): virusspecific dsRNA is also found in cells infected with single-stranded RNA viruses. DsRNA has identified in a variety of apparently normal eucaryotic cells and is associated with the "killer" character of certain strains of Saccaromyces cerevisiae.

Animals↗
PubMed1995

Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast.

The killer toxin K28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-stranded RNA, M-dsRNA (M28), that is present within the cell as a cytoplasmically inherited virus-like particle (VLP). For stable maintenance and replication, M28-VLPs depend on a second dsRNA virus (LA), which has been shown to encode the major capsid protein (cap) and a capsid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K28 toxin-coding M28-VLPs were isolated, purified and used in vitro for the synthesis of the single-stranded M28 transcript, which was shown to be of plus strand polarity and to bind to oligo(dT)-cellulose, indicating that M28(+)ssRNA contains an internal A-rich tract. Strand separation of the 1.9 kb M28-dsRNA and direct RNA sequencing of its 3' ends was performed in order to obtain specific DNA oligonucleotides that could be used as primers for cDNA synthesis. The nucleotide sequence of the toxin-coding M28-cDNA identified a single open reading frame (ORF) coding for a polypeptide of 345 amino acids, which contained two potential Kex2p/Kex1p processing sites and three potential sites for protein N-glycosylation. The toxin-coding cDNA was cloned and expressed in sensitive non-killer strains under the control of the yeast PGK promoter. Upon transformation, this construct conferred the complete K28 phenotype, demonstrating that both toxin and immunity determinants are contained within the cloned cDNA. In vitro translational analysis of the M28(+)ssRNA in vitro transcript identified the primary gene product of M28 as a K28 preprotoxin of 38 kDa (M-p38).

Amino Acid Sequence↗
PubMedDate not supplied

Isolation and characterization of a virus-resistant mutant of Cryphonectria parasitica.

Hypovirulent strain NB58 of Cryphonectria parasitica contains a dsRNA virus with a genome size of approximately 12.5 kb. Although NB58 is very stable in culture, a phenotypically-distinct sector arose which was found to be dsRNA-free. Attempts to infect the mutant strain, termed NB58F, by pairing with the parent strain (NB58) or other conversion-compatible, virus-containing strains have been unsuccessful. DNA fingerprint analysis showed that NB58, NB58F, and a representative dsRNA-free single-conidial isolate of NB58 termed NB58-19, were isogenic. The mutant culture was phenotypically stable, and all single-conidial progeny had the NB58F morphology. NB58F was intermediate between NB58 and NB58-19 in laccase production and virulence. Pigmentation and sporulation of NB58F, however, were reduced to near the level of NB58. In mating studies, NB58F functioned only as the male in sexual crosses. The mutant phenotype (F) predominated by a ratio of 5:2 among the ascospore progeny of F-type x wild-type crosses. These data suggest the lesion is nuclear and may be associated with a chromosomal abnormality. Attempts to infect the NB58F-type ascospore progeny failed, whereas the wild-type progeny were successfully infected with strains compatible with one or the other parent at a frequency of about 34%. Hyphal anastomosis and movement of cytoplasmic material occurred when NB58F was paired with a compatible strain, suggesting that the lesion is involved in viral maintenance as opposed to initial virus infection. NB58F represents the first virus-resistant isolate of C. parasitica to be described.

Crosses, Genetic↗
PubMed1993

Nucleotide sequence relationships of double-stranded RNAs in flax rust, Melampsora lini.

Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1-A5, and B1-B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near full-length sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.

Amino Acid Sequence↗
PubMed1996

Host-range restriction of vaccinia virus E3L-specific deletion mutants.

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus sigma 3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

Animals↗
PubMed1992

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis.

Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified in S. crataegensis strains Y-5910 or YB-192. Four S. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules of S. crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora. In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Blotting, Northern↗
PubMed1992

Hybridization relatedness of Israeli and U.S. bluetongue (BLU) serotypes using cDNA probes from BLU virus strain 11-UC8.

Partial cDNA clones representing 47%, 96%, and 98% of genome segments 7, 9, and 10, respectively, of a US bluetongue virus (BLU) 11 virulent strain were used to study, for the first time, the genetic relationships between Israeli BLU proto-serotypes and field isolates, and US BLU proto-serotypes. Their usefulness as group-specific identification probes was also determined. The viral nucleic acid was extracted from the infected cells and the purified dsRNA genome segments were fractionated by polyacrylamide gel electrophoresis, transferred to a nylon membrane and hybridized to the 32P labeled DNA probes. The three probes recognized all the samples tested. Genome segment 7, that code for the mayor inner capsid protein VP7, showed the most variation in the hybridization signal with the US proto-serotypes and all the Israeli samples studied. The genome segments 9 and 10 that code for the minor inner capsid protein VP6 and the nonstructural protein NS3, respectively, were highly conserved in all the samples tested despite their distant geographical regions of origin. The last two mentioned clones showed to be good group-specific probes for the identification of BLU samples from Israel and United States. The obtained cloned genetic probes were also tested against US epizootic haemorrhagic disease virus (EHDV) serotype 1 and 2 viral dsRNA, a distantly related orbivirus. None of them hybridized with the viral dsRNA of these two viruses.

Animals↗
PubMed1988

Multiple populations of double-stranded RNA in two virus-harbouring strains of Trichomonas vaginalis.

The existence of six dsRNA segments of Trichomonas vaginalis virus was confirmed and the molar mass and relative abundance of these segments were determined by agarose gel electrophoresis with reovirus dsRNA serving as a standard. The M's were 3.5, 3.4, 3.2, 2.5, 1.4 and 0.34 Mg/mol for the two strains studied, the relative abundances, however, were 1.0, 1.4, 3.0, 0.3, 2.7, 4.2 and 1.0, 0.6, 1.7, 0.5, 3.4 1.0 for these strains, respectively. Cell homogenate fractionation showed that all dsRNA segments were associated with viral particles. The data appeared to support the hypothesis of a relationship between viruses of the protozoan T. vaginalis and of the yeast Saccharomyces cerevisiae.

Animals↗
PubMed2004

RNA interference: potential therapeutic targets.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.

Animals↗
PubMed2001

Common multiple dsRNAs are present in populations of the fungus Discula destructiva originating from widely separated geographic locations.

DsRNAs were detected in 85/108 isolates of Discula destructiva, the cause of dogwood anthracnose, collected in South Carolina, Idaho, and Alabama. The eastern isolates contained a greater diversity of dsRNA than did Idaho isolates, but most isolates, irrespective of state of origin, contained two small bands (ca. 1.5-2.5 kb) with sequence homology indicated by Northern hybridization. Differences in the banding patterns suggest that genetic diversity of dsRNA in D. destructiva is generated rapidly and that D. destructiva can be simultaneously infected by multiple dsRNA viruses.

Ascomycota↗
PubMed1996

Variability and inheritance of double-stranded RNA viruses in Phaffia rhodozyma.

The present survey demonstrates polymorphism in both the length and the number of double-stranded RNAs (dsRNAs) among six Phaffia rhodozyma strains. Strains with one-, three- and four-types of dsRNA molecules were found, while two strains proved to be dsRNA-free. Elongated icosahedral virus-like particles (VLPs) 34x26 nm in size were detected in strains carrying four- or three-types of dsRNAs. One 3.7-kb dsRNA molecule was found not to form part of the VLP genome. Transmission of the VLPs of strain ATCC 24203 was followed through the basidiospores during the sexual cycle. Cytoplasmic inheritance was observed.

Basidiomycota↗