STobRV RNA Research Articles

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.

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.

Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea ( Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [ 14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 μg/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester.

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.

Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs.

An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.

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.

Cherry leafroll virus infections are affected by a satellite RNA that the virus does not support

Previous research showed that tobacco ringspot virus (TobRV), a member of the nepovirus group, acts as a supporting virus for the 359-nucleotide residue satellite tobacco ringspot virus RNA (STobRV RNA), resulting in STobRV RNA replication and its encapsidation in TobRV coat protein. In some hosts STobRV RNA decreases the yield of TobRV and the severity of TobRV-induced symptoms. We report here that inclusion of STobRV RNA in an inoculum of cherry leafroll virus (CLRV), another nepovirus, prevented the accumulation of CLRV in the inoculated leaves of cowpea ( Vigna unguiculata) and caused the symptoms to be less severe than those induced when CLRV was inoculated alone. CLRV spread to, and increased in, uninoculated, developing leaves whether or not it was coinoculated with STobRV RNA. STobRV RNA was not detected in CLRV particles or in extracts of infected tissue from the coinoculated plants, indicating that CLRV does not support STobRV RNA. STobRV RNA strongly interfered with the in vitro translation of the RNAs of CLRV and of cowpea mosaic virus (CPMV). Coinoculation of STobRV RNA and CPMV had no detected effect on infections by CPMV, so inhibition of translation in vitro and of replication in vivo were correlated only for CLRV. Results from a new in vitro assay for proteolytic processing of CLRV polyproteins gave no indication of an effect of STobRV RNA on this reaction.

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.

Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA

Small satellite RNAs1–5 of plant viruses depend on the presence of a supporting RNA virus for their propagation in vivo. Replication of the 359–nucleotide-long6 satellite RNA of tobacco ringspot virus (STobRV RNA) is detected only in tissue infected with tobacco ringspot virus (TobRV). STobRV RNA becomes encapsidated in TobRV coat protein and acts as a parasite of TobRV, reducing its accumulation and the severity of symptoms that it induces. Here we report the transcription in vitro of a circularly permuted, complementary DNA clone of STobRV RNA oriented so as to produce RNA that is complementary to the encapsidated, (+) polarity STobRV RNA. Like STobRV (+)RNA7, this dimeric, circularly permuted STobRV (−)RNA cleaves autolytically. Cleavage is at two identical sites generating monomeric-length RNA and two terminal fragments. The new termini are 5′-hydroxyl and 2′:3′-cyclic phosphodiester groups. The RNAs ligate spontaneously to give linear and, from the monomers, circular molecules. Replication of STobRV RNA may require these autoly-sis and ligation reactions, which, at least in vitro, occur without enzyme 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.

Nucleotide sequence of satellite tobacco ringspot virus RNA and its relationship to multimeric forms

Tobacco ringspot virus, a member of the nepovirus group, supports the increase and encapsidation of coinoculated satellite tobacco ringspot virus RNA (STobRV RNA). The nucleotide sequence of the unit length STobRV RNA, found here to be 359 nucleotide residues for the budblight strain, occurs also in multimeric, repetitive sequence forms. These are able to undergo an autolytic processing reaction to generate biologically active, unit length STobRV RNA (G. A. Prody, J. T. Bakos, J. M. Buzayan, I. R. Schneider, and G. Bruening,1984, In “Abstracts of the 3rd Cold Spring Harbor RNA Processing Meeting, May 16–20,1984,” p. 8). We determined the nucleotide sequence of the monomeric STobRV RNA by combining results from partial enzymatic digestions of the RNA, partial chemical cleavage of cDNA transcribed from the RNA, and analyses of cDNA clones. Other analyses gave the terminal residues of monomeric STobRV RNA: a cytosine-2′:3′-cyclic phosphodiester and a 5′ terminal adenosine. The terminal residues of monomeric RNA and their adjacent nucleotide sequences are consistent with the sequence in the junction region of dimeric RNA, derived from transcripts and cDNA clones, and with the formation of two monomeric STobRV RNAs upon autolysis of dimer, without the gain or loss of a nucleotide residue.

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.