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

Abstract

(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.