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At least 1,333 records · Page 74Linked to original sources
PubMed1986

A misaligned double-stranded RNA, poly(I).poly(C12,U), induces accumulation of 2',5'-oligoadenylates in mouse tissues.

2',5'-Oligoadenylate synthetase was induced 3-2000-fold in spleen, liver, kidney and brain of NIH Swiss mice injected intravenously with 2-200 micrograms of the misaligned dsRNA, poly(I).poly(C12,U). Levels of 2',5'-oligoadenylates extracted from these tissues were also elevated, although the amount of 2',5'-oligoadenylates extracted did not correlate directly with the amount of enzyme present. These results suggest that double-stranded portions of the misaligned polymer survived intracellularly and activated the 2',5'-oligoadenylate synthetase, and that the level of dsRNA may contribute to the control of 2',5'-oligoadenylate metabolism.

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS.

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.

Base Sequence↗
PubMed1991

Temporal regulation of tobacco mosaic virus-induced phosphorylation of a host encoded protein.

The in vitro and in vivo phosphorylation of a plant encoded protein (p68) associated with dsRNA-dependent protein kinase activity was stimulated at specific time intervals following infection by tobacco mosaic virus or electroporation with dsRNA. The level of p68 phosphorylation in infected and mock inoculated protoplasts did not differ significantly until 6 hr. post-infection, when the basal level of phosphorylation increased 2-3 fold in infected protoplasts. Maximum phosphorylation of p68 occurred between 8-12 hr post-infection and then declined but, at least until 72 hr. post-infection, it was significantly greater than in mock inoculated protoplasts.

Adenosine Triphosphate↗
PubMed1987

The genetic relatedness of a number of individual cognate genes of viruses in the bluetongue and closely related serogroups.

Genome segments 2, 4, 6, 7, 8, 9, and 10 of bluetongue virus (BTV) serotype 10 were cloned in pBR322. The 2926-bp S2 gene, which codes for the serotype-specific antigen, was cloned as two overlapping 2.4-kb inserts. The relatedness of cognate S2 genes among different isolates of BTV10 was investigated by hybridization, restriction enzyme mapping, and sequencing of the terminal ends. Hybridization under high stringency conditions indicated a genetic diversity between isolates of BTV10 from South Africa and the United States. This was confirmed by a comparison of the restriction map of the cloned S2 gene of a BTV10 isolate from South Africa to that of the S2 gene of the BTV10 strain of the United States which has been cloned and sequenced by Purdy et al. (1985). The part of the genome that was sequenced indicated, however, that this variation was confined to an approximately 10% sequence divergence in the coding region. Very few of the nucleotide substitutions resulted in an amino acid change. The genetic variation of cognate BTV genes within the BTV serogroup as well as among different members of closely related serogroups was also investigated. DNA probes from cloned BTV10 segments were hybridized to dsRNA from 24 different BTV serotypes. Genome segments S2 and S6 were found to be almost equally serotype specific. The stringency of the wash solutions after hybridization can be manipulated to determine an order of relatedness of different cognate genes. This was illustrated by the hybridization of a sensitive RNA probe of S7 to different BTV serotypes as well as to dsRNA from closely related orbiviruses. The results confirmed a relatedness between BTV and members of the epizootic hemorrhagic disease virus (EHDV) serogroups.

Amino Acid Sequence↗
PubMed1988

The cloning of full-length genome segments 2, 5, 6, and 8 of bluetongue virus (BTV) serotype 17 and studies of their genetic relatedness to United States BTV serotypes.

The double-stranded (ds) RNA genome segments 2, 5, 6, and 8, which encode the outer capsid proteins P2 and P5, and the two nonstructural proteins, NS1 and NS2, respectively, of bluetongue virus (BTV) serotype 17 have been cloned into pBR322. The length of the cloned genes indicates that the entire respective dsRNA genome has been cloned in each case. The four cloned genes were used as 32P-labeled probes to hybridize to PAGE-separated United States BTV serotypes 2, 10, 11, 13, and 17. Of these, the segment 6 clone was identified as being highly conserved and hybridized to all serotypes. Segment 8 was less conserved and segment 5 even less. The segment 2 clone was serotype specific. None of the probes hybridized with dsRNA from epizootic hemorrhagic disease virus (EHDV).

Animals↗
PubMed1989

Mechanism of interferon action. Activation of the human P1/eIF-2 alpha protein kinase by individual reovirus s-class mRNAs: s1 mRNA is a potent activator relative to s4 mRNA.

The ability of pure viral and cellular single-strand (ss) RNAs to activate the interferon-induced, double-stranded (ds) RNA-dependent P1/eIF-2 protein kinase purified from human amnion U cells was examined. In addition to the well-established activation of P1 kinase autophosphorylation in vitro by reovirus genome dsRNA, the P1 kinase was also efficiently activated by certain reovirus ssRNAs. The reovirus s1 mRNA was a potent activator of the kinase. By contrast, the reovirus s4 mRNA was a poor activator of the kinase. Likewise, adenovirus VAI RNA, transfer RNA, 5 S ribosomal RNA, and rabbit globin mRNA were not activators or were very poor activators of the purified P1/eIF-2 protein kinase. Analysis of hybrid ssRNAs produced between the reovirus s1 and s4 mRNAs revealed that both the 5' and the 3' portions of the s1 mRNA possessed nucleotide sequences capable of mediating kinase activation. Subsequent deletion analysis of the 5' portion of the s1 mRNA identified a 161-nucleotide region located between positions 416 and 576 which was sufficient for P1 kinase activation. Treatment of reovirus s1 mRNA transcripts with either ssRNA- or dsRNA-specific ribonucleases, but not with heat, destroyed the ability of s1 mRNA transcripts to activate the kinase. These results suggest that P1 kinase autophosphorylation in vitro may be selectively activated by individual ssRNAs in a differential manner, and that a secondary or higher-ordered ssRNA structure(s) may be important in mediating the activation.

DNA Mutational Analysis↗
PubMed1989

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↗
PubMed1989

Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles.

The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis. To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SA11-infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication. Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0.6% agarose gels (50 mM Tris-glycine, pH 8.8). This gel system was found to separate without disrupting SA11 single- and double-shelled virions and virion-derived core particles. SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virion-derived cores and intermediate of single- and double-shelled virions. SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels. The results showed that three distinct types of replication intermediates (RIs) were present in SA11-infected cells. The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins VP1, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34. A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34. The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34. Analysis of the formation and turnover of RIs in infected cells pulse-labeled with 35S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore RIs consisting of VP1, VP3, VP9, NS35, and NS34.

Animals↗
PubMed1989

Studies on the mechanism of the antiviral activity of ribavirin against reovirus.

We have examined the mechanism by which ribavirin inhibits the multiplication of reovirus. At a concentration of 12.5 microM (3 micrograms/ml) ribavirin inhibits viral multiplication, ssRNA formation, dsRNA formation, and protein synthesis by about 90%; when much higher concentrations are used for brief periods of time, the primary target of ribavirin is seen to be viral ssRNA synthesis. When the effect of ribavirin triphosphate (RTP) was tested on the in vitro transcription by cores of the dsRNA genome segments into plus-stranded RNA, elongation, that is, the formation of intact mRNA molecules, was found to be inhibited to the greatest extent; initiation was at least 2.5 times less sensitive, and cap formation and methylation were almost unaffected. The inhibition of elongation and initiation was not competitive with respect to any of the four nucleoside triphosphates. Remarkably, the transcription of plus strands into minus strands by immature reovirus particles (the replicase reaction) was insensitive to RTP. A model is proposed that envisages RTP binding to a site close to the catalytic site of the transcriptase. This binding is postulated to inhibit the helicase function of the transcriptase and lower its affinity for template RNA so that the likelihood of premature termination is greatly increased. The helicase activity is not, of course, necessary for the transcription of plus strands into minus strands, which would account for the differential sensitivity of the transcriptase and the replicase to RTP.

Animals↗
PubMed1990

Minus-strand RNA synthesis by the segmented double-stranded RNA bacteriophage phi 6 requires continuous protein synthesis.

Bacteriophage phi 6 contains three dsRNA chromosomes. Strand-separating agarose gels were used to study plus- and minus-strand synthesis in vivo and the effect of protein synthesis inhibitors. Analysis of phi 6 RNA synthesis shows low levels of all three dsRNAs and ssRNAs at 10 min, increasing label uptake into all RNAs except the large message from 20 to 60 min, and a greater abundance of medium and small messages than large mRNAs at late times. Isoconformers of the small message are synthesized throughout infection. Northern analysis suggests that large messages made early may persist to direct continuing translation of L-segment-encoded transcription and replication proteins. The time course of phi 6 minus-strand RNA synthesis in vivo, in the absence of background label in host RNAs, is reported for the first time. Label in minus strands is detected only after heat denaturation of RNA samples and appears sequentially in the small, medium, and large strands beginning at 20 min. At both early and late times, chloramphenicol arrests minus-strand synthesis rapidly and all three mRNAs accumulate. The results are consistent with the reovirus asynchronous model for dsRNA viral replication: plus ssRNAs made first are used as templates for minus-strand synthesis. They also indicate that replication protein(s) acts stoichiometrically.

Bacteriophages↗
PubMed1991

Characterization of a vaccinia virus-encoded double-stranded RNA-binding protein that may be involved in inhibition of the double-stranded RNA-dependent protein kinase.

The work described in this article identifies a vaccinia virus-encoded protein that may be involved in inhibition of the interferon-induced, double-stranded RNA-dependent protein kinase. Extracts prepared from vaccinia virus (WR strain)-infected cells contain an inhibitor of this kinase. Inhibition was reduced in extracts from which dsRNA-binding proteins had been removed by preadsorption to poly(rI).poly(rC)-Sepharose, suggesting that a dsRNA-binding protein may be involved in kinase inhibition. A single major virus-specific polypeptide of Mr = 25,000 (p25) bound to the poly(rI).poly(rC)-Sepharose. p25 was synthesized in a coupled in vitro transcription/translation system programmed with vaccinia cores, indicating that it is a vaccinia-encoded protein. Synthesis of p25 was detected at early times, by 2 hr post infection, peaked at 5 hours postinfection, and decreased during the late phase of virus replication. In the presence of cytosine arabinoside p25 synthesis did not decrease at late times postinfection. Kinase inhibitory activity accumulated with similar kinetics to p25, both in the presence and absence of cytosine arabinoside. Kinase inhibitory activity copurified with p25, through gel filtration, and Cibacron blue-affinity chromatography. Removal of p25 by precipitation with antiserum to p25 decreased kinase inhibitory activity in extracts prepared from vaccinia virus-infected cells. These results suggest that p25 may be necessary for the specific kinase inhibitory activity detected in vaccinia virus-infected cells.

Animals↗
PubMed1992

Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase.

The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.

Baculoviridae↗
PubMed1985

Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA.

The double-stranded (ds) RNA-dependent protein kinase is a 100,000-110,000 Mr complex of two interferon-induced subunits each having ATP binding sites: a 48,000 Mr protein (p48) which appears to be responsible for the phosphorylation of a 68,000 Mr protein (p68) in the presence of dsRNA. The p68 subunit once phosphorylated is converted to an active protein kinase capable of phosphorylating exogenous substrates such as the alpha subunit of protein synthesis initiation factor eIF2 or calf thymus histone. The phosphorylation of exogenous substrates is highly correlated with the degree of phosphate saturation of p68 and does not require the presence of dsRNA. Both the p68 and the p48 subunits of the protein kinase complex are purified by an immuno-affinity column containing monoclonal antibody specific for the p68 subunit.

Adenosine Triphosphate↗
PubMed1990

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

Amino Acid Sequence↗
PubMed1985

Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes.

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Animals↗
PubMed1990

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples.

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orbivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 x 10(3) TCID50 of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID50/ml). However, at 28 days p.i. (less than 1.6 TCID50/ml) BTV RNA was not detected using the PCR in either sample.

Animals↗
PubMed1994

Improved method for purification of viral RNA from fecal specimens for rotavirus detection.

An improved procedure to recover and purify double-stranded RNA (dsRNA) from fecal specimens is described. Guanidine isothiocyanate, hydroxyapatite, and cetyltrimethylammonium bromide were used to extract and purify rotavirus dsRNA from fecal specimens. The method is very efficient and easy to perform, and precludes the use of toxic substances such as phenol, chloroform, and Freon. It yields RNA free of enzymatic inhibitors, permitting its detection by reverse transcription-polymerase chain reaction assays. In addition, it was demonstrated that during initial clarification of the fecal suspension, the pellet must be washed at least twice to avoid massive losses of virus, viral protein, or viral nucleic acid retained in the solid debris.

Base Sequence↗
PubMed1994

Analysis of the double-stranded RNA genome segments among avian reovirus field isolates.

Nine isolates of avian reovirus (ARV) from both healthy birds and birds with different clinical illness and one commercially available vaccine strain were selected and characterized by analysis of the migration pattern of their genomic double-stranded RNA (dsRNA) segments following separation by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analyzed. The results show that the dsRNA segments of ARV were markedly polymorphic among isolates within the same serotype as well as among different serotypes. The results also show no correlation between electropherotype and disease state.

Animals↗