Nuclei and chloroplasts isolated from cell-free extracts of Nicotiana glutinosa L. leaves by sedimentation at 1000 g contain DNA-directed RNA polymerase activity. Whereas only traces of RNA polymerase activity were detected in fractions prepared from the 1000 g supernatant of healthy leaf extracts, RNA-directed RNA polymerase was readily detected in similar extracts from lettuce necrotic yellows virus (LNYV)-infected plants. This virus-associated enzyme activity was strongly inhibited by fractions containing membrane material from ruptured leaf cells of both healthy and virus-infected plants. Fractions with LNYV-associated RNA polymerase activity were prepared after treating the 1000 g supernatant with the non-ionic detergent Nonidet P-40. RNA polymerase activities both of cell-free leaf extract fractions and of purified LNYV preparations were rapidly lost during storage; loss of activity was more rapid in the presence of the detergent.
Results presented in the paper are discussed in relation to the difficulties encountered in demonstrating RNA transcriptase activity associated with purified rhabdovirus preparations. We conclude that to detect such enzymes it is necessary to use rapid procedures to obtain virus preparations free of contaminating cellular membranes.
BaltimoreD.,
HaunoA. S.,
StampferM.1970; Ribonucleic acid synthesis of vesicular stomatitis virus. II. An RNA polymerase in the virion. Proceedings of the National Academy of Sciences of the United States of America 66:572–576
BishopD. H. L.,
FlamandA.1975; Transcription process of animal RNA viruses. In Control Processes in Virus Multiplication95–152BurkeD. C.,
RussellW. C.
Cambridge: Cambridge University Press;
FranckiR. I. B.,
RandlesJ. W.1973; Some properties of lettuce necrotic yellows virus RNA and its in vitro transcription by virion-associated transcriptase. Virology 54:359–368
FranckiR. I. B.,
RandlesJ. W.1975; Composition of the plant rhabdovirus lettuce necrotic yellows virus in relation to its biological properties. In Negative Strand Viruses1224–242MahyB. W. J.,
BarryR. D.
London: Academie Press;
McleanG. D.,
FranckiR. I. B.1967; Purification of lettuce necrotic yellows virus by column chromato-graphy on calcium phosphate gel. Virology 31:585–591
RandlesJ. W.,
FranckiR. I. B.1972; Infectious nucleocapsid particles of lettuce necrotic yellows virus with RNA-dependent RNA polymerase activity. Virology 50:297–300
SemalJ.,
SpencerD.,
KimY. T.,
WildmanS. G.1964; Properties of a ribonucleic acid synthesizing system in cell-free extracts of tobacco leaves. Biochimica et Biophysica Acta 91:205–216
StampferM.,
BaltimoreD.,
HuangA. S.1969; Ribonucleic acid synthesis of vesicular stomatitis virus. I. Species of ribonucleic acid found in chinese hamster ovary cells infected with plaque-forming and defective particles. Journal of Virology 4:154–161