Identification of a novel structural interaction in Columnea latent viroid.

Pairwise sequence comparisons suggest that Columnea latent viroid (CLVd) may have originated from a recombination event involving Potato spindle tuber viroid (PSTVd) and Hop stunt viroid (HSVd). To examine the role of specific structural features in determining the host range of CLVd, we constructed a series of interspecific chimeras by replacing increasing portions of its terminal left and pathogenicity domains with the corresponding portions of PSTVd. Exchanges involving the left side of the pathogenicity domain led to lower rates of progeny accumulation in tomato, but one of the resulting chimeras was still able to replicate in cucumber. Exchanges involving the right side of the pathogenicity domain severely inhibited replication in tomato and appeared to abolish replication in cucumber. To identify potential interactions between nucleotides comprising the right side of the pathogenicity domain and other portions of CLVd, melting behaviors of circularized CLVd and PSTVd RNA transcripts were compared using a combination of temperature gradient gel electrophoresis and structural calculations. These analyses revealed an unexpected complementarity between the upper portion of the pathogenicity and terminal right domains of CLVd that facilitates breakdown of the rod-like native structure and formation of secondary hairpin II. Unlike secondary hairpin II, CLVd hairpin IV appears likely to act within the context of the genomic RNA.

Specific inhibition of macrophage TNF-alpha expression by in vivo ribozyme treatment.

The overproduction of the cytokine TNF-alpha is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-alpha production with ribozymes directed against TNF-alpha mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-alpha ribozymes were more effective inhibitors of TNF-alpha secretion than catalytically inactive ribozyme controls. Inhibition of TNF-alpha secretion was proportional to the concentration of ribozyme administered, with an IC50 of approximately 10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated approximately 6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-alpha secretion by approximately 50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1alpha. Ribozyme-specific TNF-alpha mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.

Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants.

We have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenic Nicotiana benthamiana plants from tombusvirus infections. To produce RNAs with authentic 5' and 3' termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1 and F2 RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformed N. benthamiana plants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses.

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.

Infectivity studies with highly infectious RNA inocula generated by ribozyme cleavage were used to compare the biological properties of three apparently nonviable mutants of potato spindle tuber viroid (PSTVd). One of these mutants (PSTVd-P) contains three nucleotide substitutions in the left terminal loop, and mechanical inoculation of tomato seedlings with RNA transcripts at levels equivalent to 10(3)-10(5) times the ID50 for PSTVd-Intermediate failed to result in systemic infection. Viable progeny containing a spontaneous C-->G change at position 4 could, however, be recovered from transgenic Nicotiana benthamiana plants that constitutively expressed PSTVd-P RNA. The initial mutations in PSTVd-P led to an overall weakening of its native structure in vitro, and the precisely-full-length molecule released by ribozyme cleavage in vivo was also unstable. Even RT-PCR analysis failed to reveal detectable amounts of circularized PSTVd-P among the RNAs isolated from uninfected plants. Predicted stabilizing effects of a spontaneous mutation at position 4 suggest that the appearance of viable progeny was dependent on a combination of events: errors by host RNA polymerase II during transcription of the mutant transgene coupled with a strong selective pressure against alterations in the native structure of PSTVd.

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Role of the variable domain in modulating potato spindle tuber viroid replication.

Potato spindle tuber viroid (PSTVd) is believed to undergo a series of specific structural transitions during replication. The variable domain of PSTVd is known to contain sequences that are important for replication/accumulation as well as one of three "premelting regions" which control breakdown of the native structure in vitro. We have examined the structural and biological effects of five single and two double nucleotide substitutions within premelting region 3 in an effort to isolate temperature-sensitive mutations affecting PSTVd replication or pathogenesis. None of these mutants replicated as rapidly as the wild type, and a variety of spontaneous sequence changes were detected in their progeny. Higher temperatures were able to partially overcome the inhibition of replication associated with a more stable secondary structure, but no well-defined temperature-sensitive PSTVd mutants were identified. Selective pressures arising from the interaction of assay temperature and structural stability in vivo appear capable of moving PSTVd populations between peaks of relatively high fitness. Depending on the exact nature and location of the mutation, selection may occur at the level of either the plus or the minus strand.

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

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.

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.

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.

Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.

Circular and multimeric forms of the satellite RNA of tobacco ringspot virus and their autolytic processing reactions are well known. They suggest replication models in which key elements are rolling circle transcription and the processing of the resulting multimeric RNA to generate the unit, 'monomeric' satellite RNA sequence. We prepared plasmids bearing two distinct sequences of the satellite RNA. Each was arranged to allow transcription of an oligoribonucleotide (r-oligo) of the polarity that is complementary to encapsidated satellite RNA. One sequence has the autolytic processing phosphodiester bond, ApG, and the other is located at a distance of about 150 nucleotide (nt) residues. The second r-oligo accomplished cleavage of the first, in a catalytic fashion. Analysis of truncated forms showed that 10 nt of the ApG junction-containing r-oligo and 46 of the endoribonucleolytic r-oligo were sufficient for recognition in the cleavage reaction. These results map the sequences involved in autolytic processing of the complementary polarity satellite RNA to two regions.

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.

Autolytic processing of a phosphorothioate diester bond.

A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles. Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues. The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA. We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA. The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules. Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate). However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation.