Jamal M. Buzayan

Autolytic Processing of Dimeric Plant Virus Satellite RNA

Associated with some plant viruses are small satellite RNAs that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.

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

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.

Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus

Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.