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© 1997 Oxford University Press 4085-4093

Core-associated non-duplex sequences distinguishing the genomic and antigenomic self-cleaving RNAs of hepatitis delta virus

Core-associated non-duplex sequences distinguishing the genomic and antigenomic self-cleaving RNAs of hepatitis delta virus

Timothy S. Wadkins, Michael D. Been*

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA

Received June 11, 1997; Revised and Accepted August 20, 1997

ABSTRACT

The two ribozymes found in hepatitis delta virus RNA form related but non-identical secondary structures and display similar cleavage properties in vitro. Three of the non-duplex elements hypothesized to contribute nucleotides to the catalytic core vary slightly in length between the two ribozymes and the differences are conserved in clinical isolates. Possible functional relationships of the core sequence elements were tested by systematically exchanging sequences between the two ribozymes. It was found that switching two of the elements (L3 and J4/2) from one ribozyme to the other reduced cleavage activity in both. On the other hand, exchanging the third region (J1/4) resulted in enhanced activity for one ribozyme and a smaller increase in activity for the other. Combining exchanges did not reveal any compensatory interactions involving these particular elements nor did a pattern emerge that would suggest an optimal combination of core sequences for a generalized HDV ribozyme. Non-compensatory behavior reinforces the idea that the non-duplex sequences may form sequence-specific contacts with duplex portions of the ribozyme, but, in addition, these data suggest that there may be selective pressures on the ribozyme sequences in the virus that are not reflected in the in vitro self-cleavage assays.

INTRODUCTION

The genomic and antigenomic RNA of hepatitis delta virus (HDV) contain self-cleaving sequences (ribozymes) which are thought to process products of rolling circle replication of the viral RNA to monomer sizes (1,2). In vitro cleavage of the RNA requires a divalent cation and yields a 5[prime]-hydroxyl group and a 2[prime],3[prime]-cyclic phosphate group (3-5), suggesting that the mechanism for self-cleavage, nucleophilic attack of the 2[prime]-hydroxyl or oxygen on the cleavage site phosphorous, is similar to the hammerhead and hairpin ribozymes. Both ribozymes from HDV are able to catalyze the self-cleavage reaction in high concentrations of chemical denaturants (e.g. 8 M urea or 18 M formamide) at 37°C in 10 mM Mg2+ (6-9). In addition, many forms of both the genomic and antigenomic ribozymes appear to cleave faster in the presence of denaturants, suggesting that these sequences assume interconverting inactive and active conformations (10,11).

The two HDV ribozymes are similar in size (7,8) and sequence and adopt very similar secondary structures (8,10,12,13) that are characterized by four duplexes (P1-P4), two hairpin loops (L3 and L4) and three joining sequences (J1/2, J1/4 and J4/2) (Fig. 1). The loops and joining sequences of the secondary structures will be referred to as single-stranded or non-duplex, even though it is likely they are involved in other interactions. The cleavage site is located at the 5[prime]-end of P1. While the secondary structures are similar, the sequences of the two ribozymes differ in the duplex regions and, to a lesser degree, in the single-stranded regions. On the basis of mutagenesis, chemical probing and crosslinking studies, L3, J4/2 and J1/4 are thought to form part of the catalytic core of the ribozyme (13-16). These sequences are presumed to serve similar roles, but they are not identical in size. The genomic L3 is 1 nt longer than the antigenomic, the genomic J1/4 is 3 nt longer than the antigenomic and the genomic J4/2 is 1 nt shorter than the antigenomic. To the extent that insertions or deletions have been made at the positions of the `extra' nucleotides, they have been shown to have small but measurable effects on cleavage activity (11,17). While such mutagenesis data might suggest that the identity of the base at these positions is not crucial for cleavage activity, conservation of the sequences in clinical isolates in which there is extensive sequence variation in other regions of the genome would suggest that these sequences are nevertheless under selective pressure (11,18,19). Thus, in contrast to the situation with the hammerhead self-cleaving RNAs, where numerous natural examples are consistent with highly conserved single-stranded (core) sequences (20), the two HDV ribozymes show some distinct differences.


Figure 1 Secondary structures of SA1-2 (antigenomic) (A) and TGR1 (genomic) (B) HDV ribozyme sequences. Nucleotide positions considered in this study are highlighted and circled. The entire sequence of each precursor is shown, the cleavage site is indicated and lower case letters at the 5[prime]- and 3[prime]-ends are non-HDV sequences contributed by the vector.

There are several possible explanations for why the distinguishing features between the genomic and antigenomic L3, J1/2 and J4/2 sequences would be maintained in clinical isolates. One is that the sequence differences reflect specific combinations of single-stranded sequences which complement each other and must be maintained for full ribozyme activity. Assuming that this interaction is independent of any particular sequence in the duplex regions, the prediction is that exchanging individual sequence elements from the two different ribozymes would have negative effects on ribozyme function, but two or more changes could have compensatory effects. Another possibility is that due to sequence-specific contacts between duplex and non-duplex regions, changes to the single-stranded sequences will result in loss of activity unless a compensatory change is made in the appropriate duplex region. A third explanation is that the differences may be necessary for aspects of viral replication unrelated to self-cleavage activity of the ribozyme. In this case the in vitro cleavage reaction may simply not reveal the significance of the differences in the core-associated sequence.

The possibility that the differences may be compensatory can be addressed by introducing changes into each ribozyme such that all of the core-associated sequence elements are systematically substituted alone and in combination with the equivalent sequences from the other ribozyme. Seven variants of each ribozyme were generated in making the individual, pairwise or triple substitutions. The effects of these mutations were interpreted to indicate that these differences do not represent simple compensatory changes in the core sequence but do not rule out the possibility that these differences are linked to sequence differences in duplex regions. Finding that a subset of the exchanges may enhance activity also leaves open the third possibility, that the differences have been conserved for reasons other than optimal ribozyme self-cleavage activity.

MATERIALS AND METHODS

Enzymes and chemicals

T7 RNA polymerase was purified by M.Puttaraju from an overexpressing clone provided by W.Studier (21). Modified T7 DNA polymerase (Sequenase) was purchased from US Biochemical (Cleveland). Other supplies were purchased from commercial suppliers. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (Department of Botany, Duke University, Durham, NC).

Plasmid construction

The version of the antigenomic ribozyme sequence used in this study was cloned into the phagemid vector pTZ18U. This sequence, pSA1-2 (8), was constructed with a synthetic version of the antigenomic ribozyme sequence inserted downstream of a T7 promoter; it is identical to the wild-type antigenomic self-cleaving sequence except for introduction of a restriction site in the sequence comprising stem-loop 4. Mutants were generated by oligonucleotide-directed mutagenesis using a uracil-containing single-stranded form of the plasmid as the template (22,23). Plasmids with mutations were identified by sequencing mini-prep DNA. Following a second round of transformation, plasmid DNA was prepared from overnight cultures and purified by CsCl equilibrium density ultracentrifugation in the presence of ethidium bromide (24). All purified plasmid DNA was again sequenced before use as templates in transcriptions.

The version of the genomic ribozyme sequence used in this study was also cloned into the vector pTZ18U. This sequence (TGR1) is a version of SD200 (25) that has been altered by oligonucleotide-directed mutagenesis (22,23) as above to eliminate all but 3 nt of the sequence 5[prime] of the cleavage site. The full sequence of the resulting ribozyme is 5[prime]-GAU-GGCCGGCAUG GUCCCAGCCU CCUCGCUGGC GCCGGCUGGG CAACAUUCCG AGGGGACCGU CCCCUCGGUA AUGGCGAAUG GGAUC, where the dash indicates the cleavage site. Deletions and base changes to this initial sequence were made as described above (8,23).

The plasmid used to produce the circular ribozyme RC1 has been described previously (26). pRC1+G was generated by mutagenesis by M.Puttaraju as described above.

Transcriptions

Plasmid DNA was linearized by digestion with HindIII (antigenomic constructs) or BamHI (genomic constructs), extracted with phenol and chloroform, ethanol precipitated and transcribed in 0.05 ml reactions containing 40 mM Tris-HCl, pH 7.5, 15 mM MgCl2, 5 mM dithiothreitol, 2 mM spermidine, ribonucleoside triphosphates at 1 mM each, 0.04 mCi [[alpha]-32P]CTP, 2.5-5 µg linear plasmid DNA and 300 U T7 RNA polymerase. Incubation was for 60 min at 37°C, EDTA was added to 50 mM, formamide to 50% (v/v) and the RNA was fractionated by electrophoresis on a 6% (w/v) polyacrylamide gel containing 7 M urea. RNA was located by autoradiography, excised, eluted and recovered by ethanol precipitation. The self-cleavage reaction requires only low levels of a divalent cation and typically genomic transcripts cleaved extensively during synthesis under the above conditions. To increase the fractional yield of uncleaved genomic RNA, 2.5 µg HindIII-linearized plasmid that when transcribed produces an RNA complementary to the 5[prime]-region of the ribozyme RNA spanning the cleavage site was added to these transcription reactions. Circular ribozymes were synthesized as previously described (26).

Cleavage assays

Radiolabeled precursor RNA was preincubated at 37°C for 10 min in the cleavage cocktail minus Mg2+ and the cleavage reactions initiated by addition of MgCl2 (37°C); final conditions were 40 mM Tris-HCl, pH 8.0, 1 mM EDTA, 11 mM MgCl2 and [sim]5-50 nM RNA. For reactions that contained 40% formamide (v/v) the formamide was included in the preincubation. The kinetics of cleavage were followed by removing and mixing 5 µl aliquots with formamide-dye mix containing EDTA. The precursor and product were separated by gel electrophoresis under denaturing conditions (6% polyacrylamide gels containing 7 M urea, 0.05 M Tris-borate, pH 8.3, 0.5 mM EDTA). The relative amounts of precursor and 3[prime]-cleavage product were quantified by analysis in a phosphorimager (Molecular Dynamics). After correcting the counts for background, the fraction cleaved (F) was calculated as countsproduct/(countsprecursor + countsproduct). The 3[prime]-fragment contained 95-97% of the label for the antigenomic precursors used in this study and 100% of the label for the genomic precursors. The 5[prime] product RNA migrates off these gels and was not included directly in the analysis because correcting the data for this small difference had no effect on values obtained for the rate constants. The first order rate constant (k) and end point (m) were obtained by fitting the data to F = m × (1 - e-kt). The end points seen in reactions with purified precursors most likely did not reflect a true equilibrium between the cleaved and uncleaved forms, since the extent of cleavage can vary for different methods of preparation of the precursor. In addition, the extent of cleavage seen with purified precursor is routinely different to the extent of cleavage observed during transcription. More likely, the end point represents a combination of contaminating species that co-migrate with the precursor during gel purification and non-cleaving conformers of the ribozymes. For several of the precursors the reaction was complete after 1 min and, because the earliest time points were taken at 4 or 5 s, values for rate constants >5 min-1 were most likely minimal estimates. Thus, while reproducibility is usually good, we may have underestimated the fastest reactions. For the precursor RNAs which cleaved more slowly, reproducibility was often within ±10%. All rate constants reported were the average of at least three independent determinations from two preparations of the same RNA.

Thermal denaturation

The thermal melting behavior of the RNA was characterized using an Aviv UV spectrophotometer. 3[prime] Product RNA(2.5 µg/ml) was degassed in a solution of 5 M urea, 1 mM EDTA, 10 mM PIPES, pH 6.5, then heated to 95°C in the cuvette with stirring. MgCl2 was added to 3 mM and the solution cooled to 25°C. UV absorbance was collected at 0.5°C intervals as the RNA was heated from 25 to 85°C at a rate of 0.5°C/min. The sample in the cuvette was stirred constantly. Data collected from three runs with fresh RNA samples each time were combined and normalized. The first derivative was obtained after smoothing the absorbance data (using KaleidoGraph software).

RESULTS

Properties of the antigenomic and genomic ribozymes

The starting sequence for the antigenomic sequence was SA1-2, which has been used in previous studies (8,12,15,17,27,28). The conditions used for cleavage in this study were 10 mM Mg2+, 40 mM Tris, pH 8.0, at 37°C in either the absence or presence of 10 M formamide. Fitting the data from a cleavage time course of the SA1-2 precursor to the single exponential form of a first order rate equation gave a rate constant of 4 min-1 (Fig. 2A and Table 1). The rate constant increased to 18 min-1 in the presence of 40% formamide. These values are close to numbers obtained in earlier studies (12,17), but, as noted previously (17), the kinetics of cleavage of this particular RNA deviated from an ideal first order reaction curve generated with the obtained rate constant and end point. A probable explanation for this is that there were two or more conformations of the precursor, resulting in one population which cleaved rapidly and one or more populations that either cleaved slowly or, more likely, must be refolded prior to cleavage. Thus, the observed rate constants reported in this case may reflect a mixture of different reactions which could include folding, refolding and cleavage chemistry. The plot of data from cleavage in the absence of formamide suggested that [sim]20-25% of the RNA cleaved prior to the first time point at 5 s, [sim]40% cleaved at a slower rate and the remainder did not cleave. Fitting the same data to an equation for the sum of two first order reactions gave a rate constant for the slower (upper) portion of the curve of 1.6 min-1 (not shown). The rate constant for the faster cleaving population cannot be accurately estimated with the current data, but 20 min-1 is consistent with a population representing 25-30% of the total precursor cleaving to 80% completion in 5 s. Of the RNA sequences used in this study, SA1-2 deviated the most from simple first order behavior. It is likely, but difficult to prove, that the 4- to 5-fold rate enhancement observed upon addition of formamide was due to disruption of inactive conformers. As will be seen, none of the other precursors used in this study demonstrated such a large rate enhancement for cleavage when formamide was added.

Table 1 . Antigenomic-derived ribozymes
Sample k (min-1)a EP (%)b kf (min-1)c EPf (%)b
SA1-2 4.0 ± 1.2 65 18 ± 1 74
[alpha]+U 3.3 ± 0.5 62 5.2 ± 0.2 77
[alpha]-G80 2.3 ± 0.5 62 0.56 ± 0.03 80
[alpha]+U-G80 1.0 ± 0.1 44 0.11 ± 0.02 52
[alpha]+CAA 23 ± 2 78 27 ± 3 84
[alpha]+CAA+U 13 ± 6 55 8.1 ± 1.9 62
[alpha]+CAA-G80 6.5 ± 1.9 11 2.5 ± 0.4 68
[alpha]+CAA+U-G80 1.7 ± 0.2 65 0.43 ± 0.02 80
A20G:U32C 0.029 ± 0.001 64 0.24 ± 0.003 72
A20G:U32C-G80 0.019 ± 0.001 62 0.010 ± 0.002 55
aCleavage in 10 mM Mg2+ at 37°C. Averaged first order rate constant and standard deviation.
bEP, end point or extent of cleavage; EPf, end point in the presence of 40% formamide.
cCleavage with addition of 40% formamide.


Figure 2 Cleavage kinetics for the antigenomic sequences. Graphs show data for SA1-2 (A) and five antigenomic variants (B-F, as indicated on the figure). Curves are fitted to the equation for a first order reaction with the rate constants and end points from Table 1. Data for reactions in the absence of formamide are indicated by circles and solid lines. Triangles and dashed lines are data generated in the presence of 40% formamide. For all graphs the fraction cleaved is plotted as a function of time in minutes; note that individual time axes vary from 1.5 to 10 min. The inset in (A) is an autoradiogram showing a representative time course from which the graphed data was obtained.

Enhancement of the rate and extent of cleavage has also been seen with some precursors containing the genomic ribozyme (6). However, the genomic ribozyme used in this study (TGR1) (Fig. 1B) is a derivative of SD200 (25) used previously. In this case the 5[prime]-end of TGR1 was shortened to 3 nt to remove non-HDV sequences 5[prime] of the cleavage site. In the absence of formamide 78% of the TGR1 precursor cleaved with a rate constant estimated to be 20 min-1 (Fig. 3A and Table 1). With 40% formamide 87% of the RNA cleaved with a rate constant of 11 min-1. Thus, unlike SA1-2, most of the TGR1 RNA appeared capable of rapid cleavage in the absence of denaturants and the addition of formamide had the more reasonable effect of slowing the reaction. It was also apparent from the plot of the data that cleavage of TGR1 more closely fits a first order reaction. Thus, with this form of the genomic ribozyme it appeared that most of the RNA folds into an active conformation and the addition of denaturants disrupted the active structure.


Figure 3 Cleavage kinetics for the genomic sequences. Graphs showing data for TGR1 (A) and five genomic mutants (B-F) as indicated on the figure. See legend to Figure 2. Rate constants and end points are given in Table 2.

Antigenomic to genomic sequence changes in L3, J1/4 and J4/2

The effect of individually changing each of the sequences in the antigenomic ribozyme was tested first. Deletion of G80 ([alpha]-G80) (Fig. 2B and Table 1) has been reported previously (17) and the results obtained in this study are similar to those results. The rate of cleavage decreased 2-fold relative to SA1-2 and addition of formamide resulted in an additional 4-fold drop in activity, which suggested that G80 contributed to stability of the ribozyme. Introducing a U to the 3[prime]-end of L3 in the antigenomic sequence ([alpha]+U), to give L3 the same sequence as the genomic ribozyme, slowed the reaction only slightly in the absence of formamide (3.3/min), but in formamide the mutation reduced the rate of cleavage 3- to 4-fold (5.2 min-1) (Fig. 2C and Table 1). Although this precursor cleaved faster in the presence of formamide, the effect was less dramatic than with SA1-2. A very different result was obtained with changes in the J1/4 sequence. Adding the CAA sequence to the J1/4 region of the antigenomic ribozyme ([alpha]+CAA) resulted in enhanced cleavage activity (Fig. 2D and Table 1). With this change [sim]80% of the RNA cleaved with a rate constant [ge]20 min-1. Assuming that SA1-2 tends to misfold, these results suggest that addition of these 3 nt to J1/4 either reduced the tendency for misfolding or greatly facilitated refolding to the active conformation.

An effect on the structure was confirmed in the thermal denaturation profile of SA1-2 and the three variants. The 3[prime] product RNA from each variant was purified and the increase in optical density at 260 nm was monitored as the temperature was raised. Melting the RNA at temperatures below 90°C required reducing the Mg2+ concentration to 2 mM (3 mM MgCl2, 1 mM EDTA) and including 5 M urea, but under these conditions SA1-2 cleaved rapidly at 37°C (data not shown). The first derivatives (change in OD260 versus change in temperature) were plotted along with relative OD260 versus temperature (Fig. 4A-D). Above 40°C there was evidence for at least two transitions with SA1-2 (Fig. 4A); the first derivative revealed one peak at 64°C and a shoulder at [sim]52°C. With the sequence variants there appeared to be a correlation between the shape and height of the shoulder and cleavage activity. The [alpha]+CAA variant (Fig. 4B) showed a very distinct peak at [sim]51°C, whereas [alpha]+U (Fig. 4C) had a less well-defined shoulder in this temperature range. It appeared that introduction of the CAA sequence into the J1/4 region of the antigenomic ribozyme accentuated this particular feature of the ribozyme, whereas addition of a U to L3 weakened this feature. This correlated well with the cleavage activity in denaturants, in that [alpha]+CAA cleaved better than SA1-2, while [alpha]+U cleaved worse. In the case of [alpha]-G80, which cleaved 10 times slower than [alpha]+U, there remained a shoulder at the lower temperature transition, but it was less defined than in the case for SA1-2, suggesting that the structure associated with it might also be destabilized. A common interpretation of such melting data is that the high temperature transition represents disruption of secondary structure and the lower temperature represents disruption of tertiary structure, but for some complex structures this is probably an oversimplification (29). Nevertheless, given that the sequence changes were made in regions defined as single-stranded in the secondary structure, these data suggest that the transition seen in the 50-53°C range could identify one or more tertiary interactions required for cleavage activity.


Figure 4 Thermal denaturation (melts) of antigenomic ribozymes. (A) SA1-2; (B-D) three single mutants indicated on the figure. Plots are of relative OD at 260 nm versus temperature (open circles). Closed circles are the first derivative ([Delta]OD/[Delta]T) after smoothing to emphasize the inflection points as peaks.

Table 2 . Genomic-derived ribozymes
Sample k (min-1)a EP (%)b kf (min-1)c EPf (%)b
TGR1 20 ± 3 78 11 ± 0.3 87
[gamma]+G 4.1 ± 0.2 77 4.0 ± 0.2 84
[gamma]-U27 12 ± 2 64 11 ± 1 81
[gamma]+G-U27 3.3 ± 0.1 68 7.2 ± 1.0 85
[gamma]-CAA 31 ± 3 74 16 ± 1 83
[gamma]-CAA-U27 25 ± 2 64 15 ± 1 83
[gamma]-CAA+G 3.8 ± 0.1 75 3.5 ± 0.2 85
[gamma]-CAA+G-U27 6.9 ± 1.0 71 7.7 ± 0.2 93
aCleavage in 10 mM Mg2+ at 37°C. Averaged first order rate constant and standard deviation.
bEP, end point or extent of cleavage; EPf, end point in the presence of 40% formamide.
cCleavage with addition of 40% formamide.

Reciprocal changes in the genomic ribozyme argue against an optimal common core sequence

In the genomic ribozyme addition of a G between A78 and U79 in J4/2 ([gamma]+G) resulted in a 5-fold decrease in activity (Fig. 3B and Table 2). No effect on the rate or extent of reaction was seen with the addition of formamide to the reaction, but it was down 2- to 3-fold relative to TGR1 under the same conditions. Deleting the U from genomic L3 ([gamma]-U27) resulted in a 2-fold decrease in rate, a lower extent of cleavage and deviation from first order behavior (Fig. 3C and Table 2). Adding formamide resulted in a small increase in the extent of the reaction but the initial rate of the reaction was about the same as in the absence of formamide and close to that of TGR1 under the same conditions. The surprising finding was that deletion of the CAA sequence in J1/4 ([gamma]-CAA) resulted in, if anything, a small increase (1.5-fold) in the rate of cleavage in both the absence and presence of formamide (Fig. 3D and Table 2). Because [gamma]-CAA cleaved extensively in the first few seconds of the reaction, it is difficult to determine if this effect is significant. However, in two other genomic sequence combinations in which cleavage rates are easier to measure, deleting CAA had the same effect: [gamma]-CAA-U27 cleaved faster than [gamma]-U27 and [gamma]-CAA+G-U27 cleaved faster than [gamma]+G-U27.

Thus these data are essentially the opposite of what the data for the antigenomic ribozyme would have predicted if either of the two ribozymes had an optimal sequence for these particular regions. For example, deleting G80 from the antigenomic ribozyme reduced activity, but so did inserting a G at the equivalent position in the genomic sequence. Likewise, deleting U27 from the genomic ribozyme reduced activity slightly, but so did inserting a U at an equivalent position in the antigenomic sequence. On the other hand, most of the effects on cleavage rates tended to be small. Only in one case ([alpha]-G80 in formamide) did the rate constant for cleavage change by >5-fold and in that case it dropped 30-fold relative to SA1-2. For comparison, effects of 102- to 104-fold were seen with mutations at several other positions in these single-stranded regions in previous studies (13,17,30).

Combinations of L3 and J4/2 sequence changes are not compensatory

The small but reproducible decrease in activity seen under some conditions with the changes in L3 or J4/2 could be explained if G80 in the antigenomic ribozyme compensated for the lack of the equivalent of the genomic U27 and vice versa. This appeared not to be the case. Combining the [alpha]+U and [alpha]-G80 mutations resulted in a greater decrease in activity than either individual change (4-fold and 16-fold in the absence and presence of formamide respectively) (Table 1). As with the antigenomic sequence, combining the changes in the genomic sequence ([gamma]+G-U27) did not restore a higher rate of cleavage (Table 2). In this case, the double mutant had a cleavage rate intermediate between the two single mutants for the reaction in formamide and that is not evidence for a compensatory interaction. Thus, the data suggested that the differences in L3 and J4/2 between the two ribozymes are not compensatory.

For completeness, other combinations of these changes were also examined (Tables 1 and 2). While some combinations which included the J1/4 change (either [alpha]+CAA or [gamma]-CAA) cleaved faster than the wild-type sequence, none cleaved any faster than the variants with the J1/4 change alone. Nevertheless, there were some noteworthy effects which appear to defy simple explanations. In particular, combining +CAA with -G80 in the antigenomic sequence resulted in a ribozyme that only cleaved to 11% completion in the absence of denaturants, but with addition of U into L3 the resulting ribozyme cleaved to 65%. With denaturants both cleaved to >65%, but the triple substitution cleaved slower than the double substitution. Finally, ribozymes with triple substitutions were always slower than the starting sequences.

Exchange of J1/4 increased cleavage activity when combined with other changes

That changes in the core sequence could enhance activity was not unexpected, but finding that essentially the opposite mutation in J1/4 of both ribozymes enhanced activity in both was surprising. It should be mentioned that while our group has seen the same result with a different version of a genomic ribozyme (25), another group reported a small decrease in activity with this deletion when measured under different conditions (30). With the change to J1/4 the cleavage rates are now faster than can be accurately measured by manual methods. Therefore, it is worth noting that with the other variants of the antigenomic ribozyme a consistent increase in the cleavage rate is seen when CAA is inserted into J1/4; adding CAA to [alpha]+U resulted in a 4-fold increase, adding it to [alpha]-G resulted in a 3-fold increase and adding it to [alpha]+U-G resulted in a 2-fold increase. Another way to look at it is that the +U and -G changes also appear to slow down the [alpha]+CAA variant. Because none of the combinations cleave faster than both of the individual variants, these data indicate that the effects of these changes should be viewed as additive rather than compensatory.

A similar, though less dramatic, effect was seen with combinations of changes in the genomic ribozyme. Deleting CAA from [gamma]+G had no effect, but with [gamma]-U and [gamma]+G-U deleting CAA resulted in a 2-fold increase in the rate of cleavage.

A trans-acting form of the ribozyme is even more sensitive to sequence variations in J4/2

RC1 is a trans-acting circular ribozyme which is a composite of sequences (Fig. 5A; 26). It has an antigenomic P1, L3 and J1/4, but a genomic J4/2, P3 and part of P2 and its activity compares favorably with other forms of trans-acting ribozymes derived from the antigenomic sequence (12,14). Under single turnover conditions, 1 µM ribozyme and trace substrate ([le]5 nM), RC1 cleaved the substrate with a pseudo-first order rate constant of 0.6 min-1. Introduction of a G in J4/2 to make it antigenomic-like resulted in a ribozyme (RC1+G) that is essentially inactive (Fig. 5B). This rather dramatic effect suggests that these sequence differences may have much larger effects in some contexts than others and need to be taken into consideration in the construction of trans-acting ribozymes derived from the HDV self-cleaving sequence.


Figure 5 Insertion of a G80 equivalent into the trans-acting ribozyme RC1 destroys activity. (A) Secondary structure of the trans-acting circular ribozyme RC1 base paired with an 8 nt substrate. The position of the G insertion in RC1+G is indicated. (B) Graph of kinetic data for RC1 (triangles) and RC1+G (circles). Curves are fitted to the equation for a first order reaction. Numbers in parentheses are the extent of cleavage for each ribozyme.

Sequence differences in a duplex region

It is likely that the non-duplex elements form contacts with duplex sequences and some of the sequence specificity that is seen is required to maintain these contacts. One potential example with J4/2 and P3 is suggested from the above result: if P2 and P3 form a coaxial helix, the 3[prime]-end of J4/2 would be close to P3 and specific contacts would be possible. In a 3-dimensional model of the genomic ribozyme, Tanner et al. (30) have modeled A77 and A78 close to the minor groove of P3. P3 in the antigenomic ribozyme contains an A-U pair (A20-U32) at the same position as a G-C pair in the genomic ribozyme (G17-C30). In both ribozymes the data support the base pairing requirement in P3 but also reveal that the sequence of this region is required for full activity of the ribozymes (12,13). The possibility that was considered here was that the identity of this base pair co-varied with the presence of G80 in the antigenomic and with its absence in the genomic J4/2. This would partially explain how deleting G80 from the antigenomic ribozyme or inserting a G into J4/2 of the genomic ribozyme might reduce activity. Some form of interaction with G80 would also be consistent with chemical modification data on the antigenomic sequence, which indicates that G80 is protected from both kethoxal and CMCT modification (A.T.Perrotta, I.-h.Shih and M.D.Been, unpublished data).

This hypothesis was tested by converting the antigenomic P3 to the genomic version by replacing the A-U pair with a G-C (A20G:U32C). Activity dropped by >100-fold (Table 1). If G80 formed an unfavorable contact with the G-C pair, deleting G80 may then restore the higher rate of cleavage. However, combining [gamma]-G80 and the base pair swap appeared to have additive effects in that cleavage activity was reduced further. While this rules out one particular interaction, it does not rule out other possible interactions between J4/2 and P3.

DISCUSSION

In choosing to focus on the significance of sequence differences in L3, J1/4 and L4/2, we considered the following information. In both the antigenomic and genomic ribozymes L4 and most of P4 can be eliminated with little or no decrease in self-cleavage activity (12,31). J1/2 can be deleted entirely in a trans-acting ribozyme without eliminating cleavage activity (12,14). In contrast, alterations of certain bases in L3, J1/4 and J4/2 decreased cleavage activity (10- to 104-fold), suggesting that these regions contain essential nucleotides (13,17,30). In addition, cross-linking studies indicate that several nucleotides within L3 and J4/2 are close to the cleavage site (15,16). The data therefore suggest that these three regions contain nucleotides that contribute to formation of the active site.

To understand the possible significance of the sequence differences between the two ribozymes, seven combinations of the genomic and antigenomic core-associated sequences were constructed and tested for each ribozyme. Most of the reciprocal changes resulted in ribozymes that were at least marginally less active than the starting sequence, ruling out a common optimal core-associated sequence. In addition, combinations of these changes resulted in ribozymes that cleaved slower than either of the individual changes. However, there were changes, notably addition of CAA to the antigenomic J1/4, which resulted in sequences that cleaved to a greater extent and perhaps faster. Combining these faster cleaving variants with a slower cleaving variant generally resulted in ribozymes with intermediate rates. Thus, combinations of mutations showed additive effects suggesting that the effect of each of these changes acts independently.

A small but reproducible increase in activity was seen when the CAA sequence was deleted from the genomic sequence. We have previously considered the possibility that the J1/4 sequence is misdrawn in the secondary structure of the genomic ribozyme (10,12), in that the base of P4 could be formed from C41 pairing with G73 and A42 pairing with U72 rather than as drawn (C44-G73 and A45-U72). This would make the genomic and antigenomic J1/4 look exactly the same and is thus an attractive model. The deletion and insertion experiments here do not distinguish between the alternative pairing possibilities, but one of the clinical isolates does (19). In that case, the genomic ribozyme from the South American isolate Peru-1 (18) is not capable of forming the alternative pairing and thus provides good support for the original model with a 6 nt J1/4 (19). However, the data in this paper may also be interpreted to indicate that either arrangement at the J1/4-P4 junction would be active.

Given the small effects that most of these changes have on ribozyme activity and the seemingly idiosyncratic behavior of the mutations, one could question whether or not these sequence differences are important. However, the strongest support that these specific sequences are required remains conservation of these sequences in all of the clinical isolates (11,19). Why, for example, do all of the genomic sequences have an 8 nt L3 if the antigenomic 7 nt L3 works just as well? In fact Nishikawa et al. (32) recently showed, using in vitro selection from a mutagenized version of a trans-acting genomic-derived ribozyme, that the antigenomic form of L3 was selected. However, in the self-cleavage reaction both we and Thill et al. (1993) see a 2-fold decrease in activity when U27 is deleted from the genomic L3.

There are multiple explanations for sequence differences between the two ribozymes which are consistent with the effects seen with the exchanges. First, it is very likely that there are sequence-specific interactions between the single-stranded regions which were varied and other parts of the molecule, particularly the duplex regions defined in the secondary structure. However, except for the possible G·G pair at the end of P4 (25), no other examples of such contacts have been identified. The second explanation stems from the uncertainty and problems involved in defining a ribozyme that is naturally part of a larger molecule and defining the conditions under which cleavage should be assayed. Thus, the isolated ribozyme sequence no longer reflects the normal situation and the in vitro assay for cleavage of neither ribozyme as defined in vitro is optimal for cleavage activity. It is also possible that sequence differences could reflect a need for regulation of the ribozymes, either in relation to the asymmetry in the replication process which results in genomic RNA in 20-fold greater abundance than antigenomic RNA or in synthesis and polyadenylation of the mRNA for the [delta] antigen (33,34). Although it has been difficult to demonstrate in vitro, there is now evidence that a protein coded by the virus, the [delta] antigen, facilitates RNA cleavage activity in cells (35).

For applications of ribozymes, especially in trans, the data indicates that cleavage activity can be either inhibited or enhanced with simple sequence alterations to the core-associated regions. Unfortunately, no unique combination for optimal activity was apparent. Although this would at first appear to be a major complication to generating trans-acting ribozymes targeted to specific sequences, it may actually provide a route for modifying ribozyme sequences such that a greater variety of substrate sequences could be targeted at high specificity.

ACKNOWLEDGEMENTS

We thank M.Puttaraju for plasmids pRC1 and pRC1+G, A.Perrotta for assays on the trans-acting ribozymes and S.Carty for performing the melts on the 3[prime] product RNAs. We appreciate comments on the manuscript from I.-h.Shih, A.Perrotta and G.Wickham. This work was supported by a grant from the NIH (GM-17233).

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*To whom correspondence should be addressed. Tel: +1 919 684 2858; Fax: +1 919 684 5040; Email: been@biochem.duke.edu
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