A nucleotide sequence rearrangement distinguishes two isolates of satellite tobacco ringspot virus RNA.

Several strains of tobacco ringspot virus (TobRV) support the replication and encapsidation of satellite tobacco ringspot virus RNA (STobRV RNA). We have compared the nucleotide sequences of four STobRV RNAs, each initially associated with a different isolate of TobRV. A STobRV RNA from a geranium isolate of TobRV and STobRV RNA from the previously analyzed budblight isolate (J.M. Buzayan, W.L. Gerlach, G. Bruening, P. Keese, and A.R. Gould, 1986, Virology 151, 186-199) differed by a single nucleotide residue substitution. STobRV RNAs from TobRV isolates 62L and NC-87 have the same 360-residue nucleotide sequence. This sequence differs from that of the 359-nucleotide residue budblight STobRV RNA principally at locations 100 through 140. The differences between the two sequences in this region are consistent with a rearrangement of blocks of nucleotide residues. The two sequences can be folded with similar patterns of base pairing. All four STobRV RNAs share a sequence of eighty 5'-terminal and of twenty 3'-terminal residues, including the 5' hydroxyl group and 2':3'-cyclic phosphodiester group.

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.

Autolytic processing of dimeric plant virus satellite RNA.

Associated with some plant viruses are small satellite RNA's 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.

Multimeric forms of satellite of tobacco ringspot virus RNA.

An approximately 350-nucleotide residue RNA replicates in association with tobacco ringspot virus (TobRV) and becomes encapsidated in TobRV coat protein. Here we show by electrophoretic analyses that this small satellite RNA, RNA S, is the most abundant and most rapidly migrating of a series of at least ten encapsidated RNAs with RNA S sequences. A largely double-stranded RNA fraction from infected tissue, when denatured, gave a similar series of up to 12 zones that contained both RNA S sequences and sequences that hybridized to RNA S. Analysis of the mobilities suggests a weight increment between each zone corresponding approximately to the size of RNA S. Thus the more slowly migrating zones appear to contain covalent multimers of RNA S or, for tissue RNA, both multimers of RNA S and multimers of the complement of RNA S sequences. Neither terminal structure of TobRV genomic RNAs was found in the satellite RNA. RNA S lacks detectable polyadenylate or oligoadenylate. Covalently linked protein was not detected in RNA S or its more slowly migrating forms, and satellite RNA biological activity, unlike that of the TobRV RNAs, was not protease sensitive. Polynucleotide kinase catalyzed the phosphorylation of satellite RNAs, indicating free 5'-hydroxyl groups.

Electron microscopy of double-stranded nucleic acids found in tissue infected with the satellite of tobacco ringspot virus.

Electron microscopic studies of the ds RNAs purified from plants infected with the satellite (S) of tobacco ringspot virus (TobRSV) revealed that approximately 91% of the population is linear, varying in length between 40 and 3000 nm. Six percent of the population appeared as relaxed circles, and the balance appeared as "racket"-like structures. Electrophoretic analysis of these preparations detected at least 12 components higher in molecular weight than the expected 230,000-dalton RF. Denaturation of the same sample released ss RNA that coelectrophoresed with STobRSV RNA from virions. The linear ds molecules, although extremely variable in length, have some preferential distribution around 130 nm, not around 68 nm, the length of ss STobRSV RNA; the circles also varied, but their lengths fell into four distinct peaks: the shortest was 130 nm, and the increment of each longer circle was also about 130 nm. The circular portion of the "racket"-like structure was a uniform 130 nm, with a varied linear portion. However, the most frequent linear dimension was also 130 nm. Denatured ds RNA varied in length, and most molecules fell in between 50 and 185 nm. Relaxed circles and "racket"-like structures were also present, but much less frequently than in the undenatured preparation. T1 RNase degradation of ds StobRSV RNA made the linear population more uniform in length (between 60 and 130 nm), most preferentially around 130 nm, and decreased the number of circles and "racket"-like molecules.

Satellite-like particle of tobacco ringspot virus that resembles tobacco ringspot virus.

A satellite-like nucleoprotein serologically indistinguishable from multicomponent tobacco ringspot virus, also resembles it in size, shape, and electrophoretic mobility. Although the protein shell of the nucleoprotein is similar to, if not identical to that of tobacco ringspot virus, each ahell of the nucleoprotein conntains many small strands of nucleic acid, which replicate only in mixed infections with the virus.