Production Of Double-stranded RNA Research Articles

Characteristics of single- and double-stranded RNA synthesis by a rotavirus SA-11 mutant thermosensitive in the RNA polymerase gene.

The phenotype of a rotavirus SA-11 mutant, ts C, which carries a mutation in the gene coding for the viral RNA polymerase was studied in vitro. ts C viral transcription proved to be sensitive to temperature in a different way to that previously described. Like the wild type, the ts C mutant has an optimum for in vitro transcription at 45 degrees, but mRNA synthesis was inhibited at temperatures over 50 degrees. This mutant also showed a higher resistance to transcriptional inhibition by nucleotide analogues than the wild-type strain. The in vitro minus-strand RNA synthesis catalysed by ts C particles indicates that the mutant does not exhibit the expected increased sensitivity to temperatures over 31 degrees shown by the in vivo phenotype. As with plus-strand synthesis, the optimal temperature for the minus-strand synthesis assay was 45 degrees, but for temperatures over 55 degrees, the number of double-stranded RNA products was altered. Our results suggest that when in vitro plus- and minus-strand RNA synthesis in ts C and wild type are compared, the mutated VP1 motif affects both transcription and minus-strand synthesis, but in different ways. In infected cell cultures, the results also show that the phenotype associated with ts C seems to mainly affect the function of plus-strand RNA synthesis.

Production of double-stranded RNA during synthesis of bromouracil-substituted RNA by transcription with T7 RNA polymerase

Using T7 RNA polymerase we synthesized a short oligoribonucleotide containing bromouracil by in vitro transcription of a synthetic DNA template. Whereas the major transcript obtained had the expected size and was apparently homogeneous on a sequencing gel, additional analysis revealed the presence of double-stranded RNA in this preparation. As this was not observed when the same template was transcribed in the presence of uracil, we hypothesize that bromouracil promoted the apparition of double-stranded ‘parasitic’ RNA presumably by favouring priming for the RNA-dependent RNA synthesis of the T7 RNA polymerase or by facilitating an end-to-end copy mechanism.

Active complete in vitro replication of nodavirus RNA requires glycerophospholipid.

Flockhouse virus (FHV) is a member of the nodavirus group of positive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNA-dependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon addition of glycerophospholipid (GPL), this system reproducibly carries out complete highly active replication of added FHV RNA, producing newly synthesized (+)-strand RNA in predominantly single-stranded RNA form. This accounts for previously observed effects of Lipofectin (a mixture of GPL and cationic lipid) in the system. All tested neutral and negatively charged GPLs except phosphatidic acid support complete FHV RNA replication in this in vitro system, as do phospholipid extracts from uninfected and FHV-infected cells. Neither sphingomyelin, a membrane phospholipid that is not derived from glycerol, nor cholesterol supported FHV RNA replication. Testing of compounds derived from GPL shows that the ability of active GPL to support FHV (+)-strand RNA synthesis is dependent on the structures of both the head group and the acyl chains. Neither the phosphorylated head group nor the diacylglycerol lipid moiety alone supports RNA replication. The length and saturation of acyl chains strongly influence the ability of GPL to support RNA replication. Other characteristics of this in vitro RNA replication system and the possible role played by membranes and their components in FHV RNA replication are discussed.

Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2

A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.

In vitro replication and transcription of the segmented double-stranded RNA bacteriophage φ6

In vitro conditions that support viral-specific replication and transcription have been developed from Pseudomonas phaseolicola cells infected with the segmented double-stranded RNA bacteriophage φ6. Transcription activity, previously shown to occur by semiconservative strand displacement, labeled (+) strands of all three genome segments and produced all three corresponding genome length messenger RNAs. Replication activity for each of the three double-stranded RNA segments is observed. Our criteria for replication were formation of genomic length double-stranded RNA products and at least (-) strand synthesis activity. Mn 2+ and Sarkosyl together selectively inhibited transcription. Analysis of replication alone suggested that replication templates are the viral (+) messenger RNAs.

RNA-dependent RNA polymerase isolated from cowpea chlorotic mottle virus-infected cowpeas is specific for bromoviral RNA

An RNA-dependent RNA polymerase activity capable of synthesizing full length double-stranded RNA products only in the presence of bromoviral RNA templates has been isolated from cowpea chlorotic mottle virus (CCMV)-infected Cowpeas. No comparable discrete products were obtained when a nonbromoviral (cowpea mosaic virus) RNA was used as template. Heterodisperse, ribonuclease-sensitive products were obtained in reactions catalyzed by similar extracts from mock-inoculated (uninfected) plants in the presence of added CCMV RNA. The extraction procedure was identical to that used for obtaining virus-specific polymerase from brome mosaic virus-infected barley (Bujarski, Hardy, Miller, and Hall, Virology 119, 465–473,1982). Comparison of the activities of the barley- and cowpea-derived enzymes provides further evidence for our contention that a virus-encoded polypeptide is an integral component of these replicases.

A novel structure seen when foot-and-mouth disease virus-induced poly (U) polymerase acts in a cell-free system

A novel structure was seen during the cell-free synthesis of poly (U) by foot-and-mouth disease virus-induced poly (U) polymerase acting on a poly (A) template. An electron-microscopy spreading procedure using benzyldimethylalkylammonium chloride visualized a double-stranded RNA product containing sets of open regions where single-stranded poly (U) may be made. A doublestranded poly (A)·poly (U) structure resistant to RNase A is assumed to form during the initial 15 min of poly (U) polymerase action. Apparently partial unwinding of the double-stranded structure then occurs followed by synthesis of single-stranded poly (U) in the series of newly opened regions.

Ultraviolet inactivation of the midi variant of Q beta RNA: characterization of template activity.

Abstract— MDV‐1 RNA is a 218 nucleotide variant of bacteriophage Qβ RNA. Qβ replicase catalyzes the formation of a strand complementary to a single‐stranded (SS) MDV‐I template. Upon phenol extraction, the template and complementary strands become double‐stranded (DS). Polyacrylamide gel electrophoresis of the products of this reaction revealed SS RNA, DS RNA, and discrete intermediate bands. UV irradiation of the template caused a decrease in DS RNA production which followed single‐hit kinetics with a quantum yield of 1.6 × 10‐‐3. Concomitant with this diminished DS RNA production were increases in SS RNA and intermediate sized RNA. The latter was shown to consist of a full sized SS template annealed to a partially completed nascent strand. Upon electrophoresis, these partially completed duplexes migrated in the same positions as those found in the analysis of unirradiated template, suggesting that this RNA contains replication obstruction areas in which UV lesions cause an increase in replication inhibition.

Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase

5-Bromo-UTP was found to replace UTP efficiently as a substrate for the virion-associated double-stranded RNA replicase of Penicillium stoloniferum virus PsV-S. The double-stranded RNA product of the replication reaction with 5-bromo-UTP as a substrate gave in equilibrium caesium sulphate density gradient centrifugation a single band with a buoyant density of 1.647 g/ml, consistent with that of a hybrid double-stranded RNA consisting of one brominated and one unbrominated strand. After the reaction none of the original unbrominated double-stranded RNA (buoyant density 1.606 g/ml) could be detected. It is concluded that replication of double-stranded RNA in virions of PsV-S takes place by a semi-conservative mechanism.

Chemically-induced temperature sensitive mutants of dengue virus type 2. I. Isolation and partial characterization.

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.

Synthesis of reovirus double-stranded RNA within virionlike particles

The enzyme responsible for the synthesis of reovirus double-stranded RNA (SS → DS RNA polymerase) and its double-stranded RNA product synthesized in vitro were investigated. They were studied by determining (1) the sedimentation pattern of SS → DS RNA polymerase in cesium chloride and sucrose density gradients; (2) the sedimentation pattern of the SS → DS RNA polymerase product under nondenaturing conditions in CsCl and sucrose density gradients and (3) the response of SS → DS RNA polymerase activity and its nondenatured product to treatment with chymotrypsin. SS → DS RNA polymerase activity is associated with particles which sediment in sucrose density gradients slightly slower than virions, have a density in CsCl of 1.34 g/ml as compared with 1.36 g/ml for virions and which are converted to particles with a density of 1.43 g/ml in CsCl after treatment with chymotrypsin. Double-stranded RNA synthesized by these particles in vitro is not released but remains associated with them. These results suggest that double-stranded RNA is synthesized in a viruslike particle consisting of a corelike structure to the outside of which additional protein molecules are attached.