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PubMed1990

Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress.

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.

Cells, Cultured↗
PubMed1978

Synthesis of an oligonucleotide inhibitor of protein synthesis in rabbit reticulocyte lysates analogous to that formed in extracts from interferon-treated cells.

A heat-stable, low-molecular-weight inhibitor of protein synthesis is formed on incubation of haemin-supplemented rabbit reticulocyte lysates with ATP and double-stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse L-cell extracts. The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I).poly (C) bound to an inert support. The highly purified enzyme in its stable column-bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [alpha-32P]ATP or [gamma-32P]ATP as substrate. The radioactive inhibitor synthesised in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesised with extracts from interferon-treated mouse L-cells. The inhibitors from the two systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell-free system. The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesised in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar: a distinctly different effect was obtained with the haemin-controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.

Adenosine Triphosphate↗
PubMed1989

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase.

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.

Adenoviruses, Human↗
PubMed1973

Double-stranded ribonucleic acid from Mengo virus: production, characterization, and interferon-inducing and antiviral activities in comparison with polyriboinosinic-polyribocytidylic acid.

Mengo virus double-stranded ribonucleic acid (dsRNA) was obtained on a semi-industrial scale from infected cultures of BHK-21 cells grown in suspension. Yield of the extraction and purification operations was small (about 22 mg from 10(11) cells in a 100-liter culture). Physicochemical characterization of this dsRNA gave an estimated molecular weight close to 4 x 10(6), a density of 1.59 (similar to that of the poliovirus dsRNA), and a thermal transition midpoint of 94 C. This product was a little more toxic for the mouse, by the intravenous route, than polyriboinosinic . polyribocytidylic acid (poly I:C) and strictly comparable in this respect to poliovirus dsRNA. The interferon-inducing capacity in the mouse and the antiviral activities in the mouse (infected with encephalomyocarditis, Semliki Forest, influenza, foot-and-mouth disease, and murine hepatitis viruses) and in the rabbit (Shope fibroma virus) of the ultraviolet light-inactivated product were practically identical, on a quantitative basis, with those of poly I:C. In vitro and in vivo experiments showed the dsRNA from Mengo virus to be slightly but significantly more resistant than poly I:C to the inactivating effect of human serum.

Animals↗
PubMed1977

Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae.

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.

Cell Division↗
PubMed1979

Mak mutants of yeast: mapping and characterization.

Killer strains of Saccharomyces cerevisiae are those carrying a 1.5 x 10(6)-dalton double-stranded (ds) ribonucleic acid (RNA) (M) in virus-like particles and secreting a protein toxin. Most yeast (koller or not) also carry a 3 x 10(6)-dalton dsRNA (L). We have mapped mutations in eight of the chromosomal genes needed for maintaining M (mak genes). The mak genes are widely distributed on the yeast map, with no multigene complexes. We show that mutants defective in these and other mak genes lose M dsRNA, but not L dsRNA. The mak3-1 mutation results in markedly decreased cellular levels of L dsRNA, but mak3-1 stains do not lose L dsRNA completely. Mutation of mak16 results in temperature-sensitive growth, whereas mutations in mak13, mak15, mak17, mak20, mak22, and mak27 result in slow growth at any temperature. No effect of mak mutations on mating, meiosis, sporulation, germination, homothallism, or ultraviolet sensitivity has been found. The specificity of mak mutations is discussed.

Chromosome Mapping↗
PubMed1990

In vitro replication, packaging, and transcription of the segmented double-stranded RNA genome of bacteriophage phi 6: studies with procapsids assembled from plasmid-encoded proteins.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA (dsRNA). We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of phi 6. These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7. We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures. Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription. The particles were also capable of directly transcribing exogenous dsRNA. The replicase reactions were specific for phi 6 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles. These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

Bacteriophages↗
PubMed2006

Genome sequence, full-length infectious cDNA clone, and mapping of viral double-stranded RNA accumulation determinant of hypovirus CHV1-EP721.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

Ascomycota↗
PubMed1981

Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae.

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.

Cell Cycle↗
PubMed1982

RNA of rotavirus: comparison of RNAs of human and animal rotaviruses.

Single-stranded RNA (ssRNA) was transcribed in vitro from inner-shell particles of human rotavirus strain Wa (HRV-Wa) and a bovine rotavirus (neonatal calf diarrhea virus [NCDV]) by virion-associated RNA polymerase activity. The ssRNA product consisted of 11 RNA segments which were separated by polyacrylamide gel electrophoresis. In vitro-transcribed 32P-labeled ssRNA was used to study the genetic relatedness between rotaviruses by annealing with genomic double-stranded RNA (dsRNA) of homologous or heterologous rotavirus. All segments of HRV-Wa ssRNA were hybridized with dsRNA of HRV TK80, collected from the feces of a gastroenteritis patient, at the level of 88 to 100% of the homologous reaction. On the other hand, no segments of ssRNA from HRV-Wa hybridized with dsRNA of NCDV or simian rotavirus (simian agent 11). Similarly, ssRNA from NCDV did not hybridize with dsRNA of HRV-Wa, but hybridized with dsRNA of simian agent 11 at the level of 30% of the homologous value.

Nucleic Acid Hybridization↗
PubMed1984

Interferon-mediated, double-stranded RNA-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells.

The interferon-inducible, double-stranded RNA (dsRNA)-dependent protein kinase which phosphorylates an endogenous HeLa 69 kilodalton polypeptide or exogenous initiation factor eIF2 was inhibited during vaccinia virus infection. High interferon doses (20,000 reference units per ml) did not prevent this inhibition. The inhibition required protein synthesis but not viral DNA synthesis during infection, suggesting that an early vaccinia virus gene function was responsible. An active dsRNA-dependent protein kinase could be recovered from an inactive extract by purification on polyinosinate X polycytidylate-cellulose. An inhibitor of the protein kinase, therefore, must be present in the inactive extract. Similar results have been obtained with mouse L929 cells. At early time points of infection, the protein kinase in cell extracts required exogenous dsRNA for activity. This argues against endogenous viral dsRNA and activation of the kinase in the intact cell. At late time points of infection (when vaccinia virus dsRNA was almost certainly formed), the inhibitor of the kinase is present. Accordingly, it seems unlikely that the kinase played any role in the interferon-mediated inhibition of virus growth observed in these cells under these particular conditions.

Animals↗
PubMed1992

Cloning of noncultivatable human rotavirus by single primer amplification.

A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.

Amino Acid Sequence↗
PubMed1992

Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region.

The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity.

Amino Acid Sequence↗
PubMed1994

Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.

Most Trichomonas vaginalis isolates are carriers of the multisegmented double-stranded RNA (dsRNA) virus. In vitro polymerase assays were performed to demonstrate the RNA-dependent RNA polymerase (RDRP) activity of purified particles. Transcripts which comigrated with the dsRNAs of the virus were readily detected as synthesized products, indicating viral RDRP activity. In addition, smaller-sized dsRNA species, possibly two of approximately 700 bp (s1) and one of 500 bp (s2), were synthesized by purified virus particles of the CsCl gradient surrounding the virus peak. No cross-hybridization with either s1 or s2 and the dsRNA segments occurred, suggesting that s1 and s2 were synthesized from different templates. An RNase A protection assay revealed that the synthesized s1 and s2 polymerase products were double stranded. Furthermore, hybridization of products with strand-specific RNA of s1 generated from cDNA indicated that only one strand was synthesized in vitro. s1 and s2 were not visualized in ethidium bromide-stained agarose gels of dsRNA of infected trichomonads grown in batch cultures. However, dsRNA profiles of the same infected organisms cultivated under defined continuous-flow conditions contained readily detectable levels of s1 and s2, indicating that amplification of s1 and s2 occurred under specific environmental conditions. These newly discovered dsRNAs were not detected in all of the virus-carrying isolates. Finally, it is noteworthy that the s1 and s2 dsRNAs and the RDRP activity were not detected in trichomonal isolates without virus or in virus-negative progeny derived from virus-positive parental isolates. These data indicate the possibility of variations in the number of dsRNAs and/or of the presence of satellites in trichomonads infected with the multisegmented virus.

Animals↗
PubMed1995

Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene.

The vaccinia virus (VV) E3L gene, which encodes a potent inhibitor of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is thought to be involved in the IFN-resistant phenotype of VV. The E3L gene products, p25 and p20, act as inhibitors of PKR, presumably by binding and sequestering activator dsRNA from the kinase. In this study we demonstrate that VV with the E3L gene specifically deleted (vP1080) was sensitive to the antiviral effects of IFN and debilitated in its ability to rescue vesicular stomatitis virus from the antiviral effects of IFN. Infection of L929 cells with E3L-minus virus led to rRNA degradation typical of activation of the 2'-5'-oligoadenylate synthetase/RNase L system, and extracts of infected cells lacked the PKR-inhibitory activity characteristic of wild-type VV. The reovirus S4 gene, which encodes a dsRNA-binding protein (sigma 3) that can also inhibit PKR activation by binding and sequestering activator dsRNA, was inserted into vP1080. The resultant virus (vP1112) was partially resistant to the antiviral effects of IFN in comparison with vP1080. Further studies demonstrated that transient expression of the reovirus sigma 3 protein rescued E3L-minus VV replication in HeLa cells. In these studies, rescue by sigma 3 mutants correlated with their ability to bind dsRNA. Finally, vP112 was also able to rescue the replication of the IFN-sensitive virus vesicular stomatitis virus in a manner similar to that of wild-type VV. Together, these results suggest that the reovirus S4 gene can replace the VV E3L gene with respect to interference with the IFN-induced antiviral activity.

Animals↗
PubMed1996

Mutations in the zinc-binding motif of the reovirus capsid protein delta 3 eliminate its ability to associate with capsid protein mu 1.

Reovirus capsid protein delta 3 binds both double-stranded RNA (dsRNA) and zinc. Previous studies have revealed that the amino-terminal zinc finger is not required for the ability of delta 3 to bind dsRNA. We expressed wild-type and mutant delta 3 molecules by in vitro transcription/translation to evaluate the importance of the zinc finger for other functions of delta 3. delta 3 molecules with mutations in the zinc finger did not form complexes with capsid protein mu 1 but bound dsRNA more efficiently than wild-type delta 3 did. In contrast, a dsRNA-binding mutant was unimpaired in its ability to associate with mu 1. Studies with delta 3 fragments support these findings and indicate that sequences critical for delta 3's interaction with mu 1 lie in the amino terminus of the molecule. Our finding that mu 1 and dsRNA do not compete for identical binding sites on delta 3 has implications for its function as a translational regulator in infected cells.

Binding Sites↗
PubMed1996

Paradoxical interactions between human delta hepatitis agent RNA and the cellular protein kinase PKR.

The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.

Animals↗
PubMed1997

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

Animals↗