Satellite tobacco ringspot virus RNA: Biological activity of DNA clones and their in vitro transcripts

Abstract

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates in association with tobacco ringspot virus (TobRV), apparently by means of intermediates that are multimeric, tandem repeats of STobRV RNA ( M. C. Kiefer, S. D. Daubert, I. R. Schneider, and G. Bruening, 1982, Virology 121, 262–273) and which are capable of autolytic cleavage to produce active monomeric RNA (G. A. Prody, J. T. Bakos, J. M. Buzayan, I. R. Schneider, and G. Bruening, Science, in press). We have prepared plasmids that contain circularly permuted dimeric and trimeric cDNA forms of the 359 residue monomeric STobRV RNA sequence. The dimeric and trimeric DNA inserts contain contiguous, unpermuted monomeric and dimeric STobRV RNA sequences, respectively. Monomeric RNAs of both the encapsidated, (+), and the complementary, (-), polarities were prepared in vitro: transcripts of cloned sequences were initiated at the bacteriophage SP6 promoter, and these autolytically processed to generate RNA with the electrophoretic mobility of monomeric STobRV RNA. Monomeric (+)RNA transcripts and double-stranded DNA with a permuted trimeric sequence were biologically active, as judged by their ability to engender encapsidated STobRV RNA when inoculated to plants in the presence of TobRV. Biological activity was not detected with monometric RNA transcripts of the (-) polarity or with single-stranded DNAs that contained permuted dimeric sequences of either polarity.