Synthesis Of Double-stranded RNA Research Articles

Selective inhibition of reovirus induced RNA in L cells

We have recently described two species of viral-specific RNA that begin to form approximately 7 hours after infection of L cells with reovirus ( Kudo and Graham, 1965). One of the new RNA species was heterogenous in sedimentation properties, sensitive to low concentrations of RNase and undoubtedly single-stranded. The other species was RNase resistant and was probably the double-stranded progeny RNA. The relationship between these two RNAs is not yet known. In the present paper it is shown that puromycin, an inhibitor of protein synthesis, blocks the formation of both types of RNA when added early in the latent period. When added 9 hours post-infection, the antibiotic inhibits the synthesis of double-stranded RNA, but single-stranded RNA continues to be made.

Synthesis of double-stranded viral RNA by cell-free extracts from turnip yellow mosaic virus-infected leaves

(1) Cell-free extracts of healthy chinese-cabbage leaves synthesized RNA when incubated with the four ribonucleoside triphosphates and an ATP-generating system. (2) Cell-free extracts of chinese-cabbage leaves systemically infected with turnip yellow mosaic virus synthesized single-stranded RNA and double-stranded viral RNA when incubated with the four ribonucleoside triphosphates and an ATP-generating system. (3) The synthesis of single-stranded RNA in cell-free extracts from healthy or virus-infected leaves was inhibited by actinomycin D. The synthesis of double-stranded RNA was not inhibited by actinomycin D in cell-free extracts from virus-infected leaves. (4) The single-stranded RNA synthesized in cell-free extracts from infected leaves appeared to be degraded during the incubation. It was not possible to demonstrate synthesis of intact TYMV-RNA. (5) Addition of 2-thiouracil to the cell-free extract stimulated the incorporation of [ 3H]ATP into double-stranded viral RNA. In contrast, in intact plants 2-thiouracil inhibited the incorporation of [ 32P i] into double-stranded viral RNA when the analogue was sprayed onto TYMV-infected chinese-cabbage leaves for several days before labelling the plants through the roots. (6) In cell-free extracts prepared from infected chinese-cabbage leaves previously sprayed for 4 days with 2-thiouracil, incorporation of [ 3H]ATP into double-stranded RNA was reduced by the addition of 2-thiouracil. (7) Attempts to detect incorporation of [ 35S]2-thiouracil into TYMV double-stranded RNA in the cell-free system were unsuccessful. (8) These results do not exclude the possibility that 2-thiouracil is incorporated into double-stranded TYMV-RNA in small amounts. However, they suggest that incorporation of 2-thiouracil is not the reason for the rapid enhancement of TYMV protein shell synthesis by 2-thiouracil, although it may explain the inhibition of TYMV nucleoprotein synthesis and the apparent “jamming” of the double-stranded viral RNA after prolonged spraying of TYMV-infected leaves with the analogue.

Synthesis of Double-stranded RNA in RNA-phage Infected E. coli Cells

THE reproduction of RNA-containing bacteriophages is independent of host DNA and DNA-synthesis1–4. An understanding of the underlying processes thus should lead either to the detection of a double-stranded form of RNA, which would be an analogue to the replicative form of single-stranded DNA5, or to a novel mode of single-strand replication. To check for an RNA-intermediate of phage-RNA replication, extracts were prepared from E. coli cells infected with the RNA-containing phage ƒr (refs. 6–8), On stepwise elution9 from a methylated albumin (MAK) column10, two RNA-fractions separated which were not detected in extracts from non-infected bacteria: one fraction, eluting at 0.70 M sodium chloride, resembled the RNA extracted from purified ƒr-particles. The other fraction, eluting at 0.60 M sodium chloride, will be designated at 0.6–RNA and was prominent by its resistance towards digestion with RNase.