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J Biol Chem, Vol. 273, Issue 41, 26455-26461, October 9, 1998


Identification and Characterization of the RNA Chaperone Activity of Hepatitis Delta Antigen Peptides*

Zhi-Shun Huang and Huey-Nan WuDagger

From the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replication in vivo.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Hepatitis delta virus (HDV)1 is a subviral pathogen that requires hepatitis B virus (HBV) to supply envelope protein for completion of package, secretion, and infection (1-3). The genome of HDV is a single-stranded circular RNA of ~1700 nt and HDV RNA and is replicated through a rolling circle mechanism (4). HDV codes one protein of two forms during infection: the small delta antigen (SdAg) contains 195 aa and the large delta antigen (LdAg) has an extra 19 aa at the C terminus (5). Transfection studies with HDV cDNA elucidated that the two protein forms have distinct functions. SdAg initiates genome replication (6) and LdAg promotes package (7). There are two RNA binding domains in each protein form. The first is the arginine-rich sequence near the N terminus, and the second is the arginine-rich motifs (ARMs) located at the middle one-third of the protein (8, 9). The RNA binding activity is important for the function of the two protein forms: the second RNA binding domain of SdAg is required to initiate genome replication (9-12), and the first RNA binding domain of LdAg is responsible for potent inhibition of replication (13). The specific interactions between the hepatitis delta antigen and HDV RNA appear to be involved in the regulation of virus replication although a molecular mechanism has not yet been elucidated.

HDV RNAs of genomic and antigenomic senses cis-cleaved in the absence of protein factors in vitro (14). The ribozyme activity of HDV RNA, which requires a pseudoknot-like structure of the RNA molecule (15, 16) and the catalysis of divalent cations (17), is essential for generating monomeric size RNA molecules during replication (18). Recently, Jeng et al. (19) illustrated that hepatitis delta antigen may enhance, though is not required for, the processing of multiple length HDV RNA in vivo. Conceivably, hepatitis delta antigen per se or together with some other factor(s) acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA.

RNA chaperones are proteins that aid in the process of RNA folding by preventing misfolding or by resolving misfolded species (20). The RNA chaperone activities of several proteins that bind RNA with broad specificity have been explored through their effects on hammerhead ribozyme reactions and group I intron reactions. These proteins, including the nucleocapsid protein (NC) of human immunodeficiency virus (HIV), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 (A1 CTD), and Escherichia coli ribosomal proteins, can overcome the general limitations of ribozyme reactions, such as the formation/dissociation of base pairs and the adoption of functional structure, and facilitate ribozyme catalysis (21-24).

Here we analyze the putative RNA chaperone activity of hepatitis delta antigen peptides in vitro. Using the facilitation of trans-cleavage reactions of the previously characterized hammerhead ribozyme HH16 and its 17-nucleotide substrate S (25) as the initial assay, we identify the strand-annealing and strand-dissociation activities of hepatitis delta antigen peptides. We then show that the functional hepatitis delta antigen peptides promote RNA unfolding that stimulates interstranded duplex formation. This activity is able to activate an antisense RNA as well as facilitate trans-acting hammerhead ribozymes to find their targets in cognate substrate RNAs. In addition, hepatitis delta antigen peptides can modulate the cis-cleaving activity of HDV genomic RNA fragments. Hepatitis delta antigen acts as an RNA chaperone and the RNA chaperone domain locates at the N-terminal domain of the protein that contains a high density of basic amino acids. Our findings suggest that the RNA binding domains of hepatitis delta antigen identified previously are, therefore, not required for RNA chaperone activity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Expression and Purification of Hepatitis Delta Antigen Peptide-- The cDNA of the hepatitis delta antigen and its truncated mutants were amplified by polymerase chain reactions with synthetic oligonucleotides as primers. The polymerase chain reaction products were cloned to the NdeI and BamHI sites of vector pET15b (Novagen). The sequence of each recombinant clone was determined by DNA sequencing. The plasmids were transformed into E. coli BL21(DE3) cells for expression purpose (26). The hepatitis delta antigen and its truncated polypeptides were expressed as fusion proteins with a His tag at their N termini. In fusion proteins dAg-(1-195), NMdAg-(1-143), and NdAg-(1-88), the tag had the sequence of Met-Gly-(Ser)2-(His)6-(Ser)2-Gly-Leu-Val-Pro-Arg-Gly-Ser-His. N5dAg-(14-59), N7dAg-(24-75), MdAg-(89-143), and CdAg-(89-195) contained the His tag and a Met in front of the hepatitis delta antigen sequence.

Fusion proteins that contain the N-terminal domain of hepatitis delta antigen bound tightly to phosphocellulose resin. These proteins were eluted from phosphocellulose columns by a buffer containing 50 mM HEPES (pH 7.8), 0.2 mM EDTA, 0.9-1.2 M NaCl, and 20% glycerol. Fusion proteins MdAg and CdAg were purified on nickel columns as described by the manufacturer except that the elution buffer contained 20% glycerol (Novagen). Fractions containing the fusion proteins were frozen in liquid nitrogen and stored at -70 °C. Protein concentration was determined by the Bradford assay. The absorbency at 595 nm from bovine serum albumin was used to establish a standard curve from which the concentration of each purified protein was determined. The yield of purified protein was ~0.2 mg from 50 ml of culture.

Peptide K7 was synthesized by peptide synthesizer using standard solid phase methods. The peptide was purified by high performance liquid chromatography, and the concentration was determined by ninhydrin assay.

Constructs and RNA Synthesis-- The 17-nucleotide S (see Fig. 2A) was made by solid-phase chemical synthesis. HH16 was synthesized by T3 RNA polymerase with synthetic DNA as template (27). HDV genomic RNA fragments were run-off transcripts of polymerase chain reaction-amplified templates. Other RNAs were run-off transcripts of linearized plasmids. The 5'-end labeled carrier-free S was prepared by incubating the RNA fragment with T4 polynucleotide kinase and excess amounts of [gamma -32P]ATP. Other RNAs were internally labeled by incorporating [alpha -32P]CTP in run-off transcription reactions. The synthetic RNAs were purified on polyacrylamide-7 M urea gels and were ethanol precipitated after being eluted from gels. RNA fragments were resuspended in TE buffer (10 mM Tris-HCl, pH 8, and 0.1 mM EDTA) before use. The concentrations of nonradioactive RNA fragments were determined by assuming a residue extinction coefficient of 260 nm of 6.6 × 103 M-1 cm-1. The concentration of labeled RNA fragments was calculated from the radioactivities of the fragment and the specific activity of the labeled NTP.

DNA fragments dS (5'-CTAGT GGGAA CGTCG TCGTC GCT-3'), drS (5'-CTAGA GCGAC GACGA CGTTC CCA-3'), dHH16 (5'-GATCG CGATG ACCTG ATGAG GCCGA AAGGC CGAAA CGTTC CCG-3'), and drHH16 (5'-GATCC GGGAA CGTTT CGGCC TTTCG GCCTC ATCAG GTCAT CGC-3') were used to generate recombinant plasmids. Plasmids pET15bS, pKSS, and pPRP19S had one copy of dS/drS duplex inserted to the XbaI site of vectors pET15b (Novagen), pBluescript KS(+), and pPRP19 (a plasmid containing the coding sequence of yeast PRP19 gene), respectively. Plasmid pKSS3 had three copies of dS/drS duplex inserted to the XbaI site of vector pBluescript KS(+). 15bS and KSS3 were XbaI run-off transcripts of T7 RNA polymerase. PRP19S was a HaeII run-off transcript of SP6 RNA polymerase. 15bS, KSS3, as well as PRP19S contained the sequence of S. rKSS was an XbaI run-off transcript of T3 RNA polymerase, and contained a copy of the complementary sequence of S. Plasmids pKSR and pKSR4 had one and four copies, respectively, of dHH16/drHH16 duplex inserted to the BamHI site of vector pBluescript KS(+). KSR and KSR4, and 28aNR5 were BamHI run-off transcripts of T7 RNA polymerase, and each contained the sequence of HH16.

Hammerhead Ribozyme Trans-cleavage Reactions-- All trans-cleavage reactions were carried out in 40 mM Tris-HCl (pH 7.5), 12 mM MgCl2, 100 mM NaCl, 0.02 mM EDTA, and 2% glycerol at 25 or 37 °C unless otherwise noted. In addition, the NaCl, EDTA, and glycerol of the reaction were from the stock of hepatitis delta antigen peptides, whereas Tris-HCl/MgCl2 was used for the trans-cleavage reaction. Typically, 50-µl trans-cleavage reactions were performed, and the cleavage of the substrate RNA by the ribozyme RNA was followed by removing ~3-µl aliquots at specific times. Further trans-cleavage was quenched by the addition of 10 µl of stop solution containing 50 mM EDTA, 7 M urea, 0.005% xylene cyanol, and 0.005% bromphenol blue. The cleavage products were resolved from substrate and ribozyme RNAs on a polyacrylamide-7 M urea gel. The fraction of uncleaved substrate at each time point was determined by quantification using a PhosphorImager (Molecular Dynamics).

The multiple turnover reaction contained 75 nM S and 5 nM HH16. S and HH16 were preannealed by heating two RNAs together at 95-100 °C for 1.5 min, and the mixture was cooled to 25 °C for at least 5 min. Within 0.5 min, hepatitis delta antigen peptide was added, and the cleavage reaction was initiated by the addition of Tris-HCl/MgCl2 solution. In general, the single turnover reactions were carried out with 0.5 nM substrate RNA and 5 nM ribozyme RNA. Two RNAs were heated separately at 95-100 °C for 1.5 min and then cooled to 25 or 37 °C for at least 5 min. The RNA solutions, the hepatitis delta antigen peptide, and the Tris-HCl/MgCl2 solution were mixed within 0.5 min to initiate the cleavage reaction.

Cis-cleavage Reaction of HDV Genomic RNA Fragments-- RNA was denatured at 95-100 °C for 1.5 min and then cooled to 37 °C for at least 5 min. RNA was incubated with or without hepatitis delta antigen peptide in 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.02 mM EDTA, and 2% glycerol at 37 °C for 30 min before the initiation of cis-cleavage by the addition of 12 mM MgCl2.

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Hepatitis Delta Antigen Peptides-- Hepatitis delta antigen peptides were over-expressed in and were purified from E. coli as fusion proteins (Fig. 1A). dAg contains the full-length small delta antigen, and NMdAg contains the first 143 amino acids of the hepatitis delta antigen. dAg and NMdAg were degraded during their purification and storage (Fig. 1B), and without further purification, the partially degraded peptides were used for the assays in this study. Fusion proteins containing the N-terminal domain (NdAg (aa 1-88), N5dAg (14-59), N7dAg (24-75)) and the C-terminal domain (CdAg (aa 89-195)), as well as the middle one-third of the hepatitis delta antigen (MdAg (aa 89-143)), were relatively stable, and each of them could be purified to near homogeneity (Fig. 1B, and data not shown). The nonspecific RNA degradation caused by the nuclease contaminant of each hepatitis delta antigen peptide was negligible at 25 and 37 °C, the temperatures at which all of the reactions of this study were carried out.


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  		Fig. 1.   The hepatitis delta antigen peptides of this study. A, schematic diagram of the peptides. The sequence of tag is shown under "Experimental Procedures." 1, 88, 89, 143, 144, and 195 correspond to the number of amino acids of small delta antigen. The sequence of aa 1-88 is shown, and the residues of the RNA chaperone domain identified in this study are underlined. B, photograph of the Coomassie Blue-stained 15% SDS-polyacrylamide gel analysis of different peptides. NdAg, NMdAg, and dAg were purified as described ("Experimental Procedures"); MdAg and CdAg of this gel were purified from a phosphocellulose column instead of a nickel column; and 10 µg of protein was used for each lane.

Effect of Hepatitis Delta Antigen Peptides on the Trans-cleavage Reaction of S and HH16-- The reactions of hammerhead ribozyme HH16 and its 17-nucleotide substrate S (Fig. 2A) characterized by Hertel et al. (25) were used as the model system to study the effect of hepatitis delta antigen peptides on RNA-mediated processes.


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  		Fig. 2.   Effect of hepatitis delta antigen peptides on the reactions of hammerhead ribozyme HH16. A, ribozyme HH16 complexed with its 17-nucleotide substrate (S). Cleavage of S by HH16 yields two products, P1 and P2. B, multiple turnover assay. The cleavage of 75 nM S by 5 nM HH16 in the presence of 0 ng/µl (open circle ), 3 ng/µl (bullet ), or 12 ng/µl (black-triangle) of dAg, NdAg, or CdAg at 25 °C. The turnover number, [product]/[HH16], was plotted versus reaction time. C, single turnover assay. The cleavage of 0.5 nM S by 5 nM HH16 in the absence of protein (open circle ), or in the presence of 3 ng/µl of dAg (black-square), NdAg (black-triangle), SDS-inactivated dAg (), or SDS-inactivated NdAg (bullet ) at 25 °C. Hepatitis delta antigen peptides were boiled in 0.1% SDS for 10 min for the complete elimination of their activity. D, the outline and the result of pulse-chase experiment. 1 nM 5'-labeled S (*S) and 10 nM HH16 were mixed and were incubated for varying times (t1) in the absence of protein (open circle ), in the presence of 3 ng/µl dAg (black-square), or in the presence of 3 ng/µl MdAg (black-triangle). After incubating at 25 °C for varying times (t1), 27 µl of 50 nM unlabeled S in 40 mM Tris-HCl (pH 7.5), and 12 mM MgCl2 was added to the 3-µl reaction mixture. The high concentration (~50-fold) and larger volume (~9-fold) of the unlabeled S would quench the further binding of *S to HH16. The cleavage of the bound *S catalyzed by HH16 was allowed to proceed for another 20 min, then the trans-cleavage reaction was stopped by the addition of EDTA. The cleavage products were resolved from the uncleaved *S on a 20% polyacrylamide gel with 7 M urea. The fraction of bound *S that was detected as the cleavage product was plotted against t1 (the duration of pulse).

The release of cleavage products from ribozyme is slower than the chemical step in the trans-cleavage reaction of hammerhead ribozyme HH16 (25). Therefore, under the multiple turnover condition with S (75 nM) in excess of HH16 (5 nM), there was an initial burst of product formation followed by much slower product formation, and one HH16 molecule could barely be used twice in the 4-h incubation period (Fig. 2B). The addition of dAg to the reaction accelerated the dissociation of cleavage products that increased turnover, and there was an ~3-fold enhancement in turnover rate with the addition of 3 ng/µl dAg (Fig. 2B). Because dAg contains degraded peptides, the activity of NdAg and CdAg to promote the reuse of ribozyme RNA under multiple turnover condition was analyzed. The results shown in Fig. 2B illustrate that NdAg but not CdAg enhanced turnover rate. Thus, the N-terminal domain rather than the C-terminal domain of the hepatitis delta antigen is responsible for the stimulatory effect.

Under single turnover conditions with HH16 in excess of S, the cleavage of S by subsaturated concentrations of HH16 was promoted by dAg; the rate of cleavage of 0.5 nM S by 5 nM HH16 was elevated approximately 10-fold when the reaction was carried out in the presence of saturated concentrations of dAg (Fig. 2C). NdAg and NMdAg that contain the N-terminal domain of the hepatitis delta antigen also facilitated the cleavage of S by HH16. In contrast, MdAg and CdAg did not possess this activity (Fig. 2C, and data not shown). Increasing dAg or NdAg concentrations up to 30 ng/µl did not inhibit the trans-cleavage reaction (data not shown). Nevertheless, boiling the functional peptides in the presence of 0.1% SDS caused the elimination of the stimulatory effect of these peptides (Fig. 2C, and data not shown). Therefore, hepatitis delta antigen peptides may promote the single turnover trans-cleavage reactions of S and HH16. In addition, the functional motif appears to reside in the N-terminal half of the hepatitis delta antigen.

Because there is no product dissociation process under single turnover conditions, a pulse-chase experiment was carried out to further address the question of whether the hepatitis delta antigen peptide promotes the annealing of S to HH16. The results shown in Fig. 2D illustrate that dAg may accelerate the association of S and HH16 that overcomes the rate-limiting step of the trans-cleavage reaction of S and HH16. MdAg, the peptide containing the arginine-rich motif of hepatitis delta antigen (9-10) did not possess this property.

In summary, hepatitis delta antigen can overcome the limitations of the reaction of hammerhead ribozyme HH16 by facilitating not only substrate association but also product release, and these properties were similar to those of the previously identified RNA chaperones (21-24). The functional domain resides in the first 88 amino acids of the hepatitis delta antigen.

Peptide NdAg Acts as an RNA Chaperone That Facilitates the Trans-cleavage Reactions of Various Substrates and Hammerhead Ribozymes-- The effect of NdAg on stimulating a trans-acting hammerhead ribozyme to find its target was used to evaluate the RNA-unfolding and -refolding activity of this peptide. RNA fragments that had the 17-nucleotide S or the 38-nucleotide HH16 inserted in foreign sequences of different lengths were synthesized, and the trans-cleavage reactions of various combinations of these substrate and ribozyme RNAs were carried out under single turnover conditions for investigating the facilitation effect of NdAg on the reconstitution of hammerhead catalytic domain.

The substrate RNAs 15bS (~60 nt) and PRP19S (~900 nt) contained one copy of S (Fig. 3A). The cleavage of 15bS by HH16 occurred at a rate significantly lower than that of S, whereas PRP19S was hardly cleaved by HH16 (Fig. 3B). Therefore, the accessibility of the 17-nucleotide S decreases dramatically as the substrate RNA elongates. Nevertheless, when the trans-cleavage reaction was carried out in the presence of 2-3 ng/µl NdAg, 15bS as well as PRP19S (0.5 nM of each) could be cleaved to near completion by HH16 (5 nM) at a rate similar to that of S. Higher temperatures were required for the efficient cleavage of PRP19S (37 °C instead of 25 °C) (Fig. 3B, and data not shown). KSS3 (~110 nt) is a substrate RNA containing three tandem repeated S: the first copy has a C to an A substitution at the sixth residue, and the other copies are wild type (Fig. 3A). Without NdAg, none of the Ss of KSS3 were accessible to HH16 (data not shown). However, each copy of S became equally accessible to HH16 when the reaction was performed in the presence of NdAg, although the mutated copy was cleaved at a slightly slower rate (Fig. 3, A and C).


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  		Fig. 3.   Effect of NdAg on the trans-cleavage reactions of various hammerhead substrate and ribozyme RNAs. A, schematic diagram of the hammerhead substrate and ribozyme RNAs of this study. The hatched boxes represent S, the dark boxes represent HH16, and the open boxes of each RNA represent the sequences derived from the cloning vectors. KSS3 contains three copies of S, the first copy has a C to an A substitution at residue 6 of S (C6A), and the other copies are wild type. KSR4 contains four copies of R. B, cleavage of 0.5 nM S (open circle , bullet ), 15bS (, black-square), as well as PRP19S (triangle , black-triangle) by 5 nM HH16 in the absence of NdAg (the open symbols) or in the presence of 2-3 ng/µl of NdAg (the closed symbols). The reactions of PRP19S were performed at 37 °C, whereas other reactions were performed at 25 °C. The fraction of uncleaved substrate was plotted against reaction time. C, cleavage of 2.5 nM KSS3 by 25 nM HH16 in the presence of 0.5 ng/µl of NdAg at 25 °C. The cleavage products were resolved by a denaturing polyacrylamide gel and were quantitated by PhosphorImager analysis. The cleavage event occurring at each cleavage site of KSS3 was calculated and was plotted against the reaction time. D, cleavage of PRP19S by 5 nM KSR (open circle , bullet ) or 5 nM KSR4 (, black-square) at 37 °C in the absence of NdAg (the open symbols) or in the presence of 6 ng/µl of NdAg (the closed symbols). The cleavage products were resolved from substrate and ribozyme RNAs on a 3.5% polyacrylamide gel containing 7 M urea. 5'P and 3'P represent the 5' and 3' cleavage products, respectively. PRP19S is the precursor RNA and KSR4 is the ribozyme. Ribozyme KSR runs off this gel.

The ribozyme RNAs KSR (~100 nt) and KSR4 (~220 nt) had one and four copies of HH16 at their 3' termini, respectively (Fig. 3A). These two RNAs catalyzed the cleavage of the short substrate S, but they could not attack the target in longer substrate RNAs (Fig. 3D, and data not shown). However, if the substrate and ribozyme RNAs were incubated with NdAg for a very short period, i.e. < 0.5 min, before the trans-cleavage was initiated by the addition of 12 mM MgCl2, KSR together with KSR4 efficiently catalyzed the cleavage of both 15bS and PRP19S (Fig. 3D, and data not shown). Therefore, the action of NdAg on RNA molecules is very fast. Trans-cleavage did not occur when PRP19S (0.5 nM) and KSR4 (5 nM) were incubated with 3-36 mM MgCl2, and the addition of NdAg to the reaction mixture stimulated the hammerhead ribozyme catalysis. However, the stimulatory effect of NdAg on PRP19S cleavage decreased dramatically as [MgCl2] of the solution increased (Fig. 4A). This finding discloses that the inactive structures of RNA molecules are stabilized by the magnesium ion that in turn diminishes the stimulatory effect of NdAg.


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  		Fig. 4.   The trans-cleavage reaction of PRP19S and KSR4. A, PRP19S (0.5 nM) and KSR4 (5 nM) were incubated with 3 (open circle ), 12 (bullet ), 24 (triangle ), or 36 mM MgCl2 (black-triangle) at 37 °C for 10 min. Then 6 ng/µl NdAg was added (as indicated by the arrow), and trans-cleavage reaction was carried out for another 20 min. The fraction of uncleaved PRP19S was plotted against the trans-cleavage reaction time. B, PRP19S (0.5 nM) and KSR4 (5 nM) were incubated with 6 ng/µl of NdAg at 37 °C for 10 min. NdAg was removed by phenol/chloroform extraction within the next 10 min. Trans-cleavage reaction was initiated and was carried out in the presence of 12 mM MgCl2 for 10 min (black-triangle). Then either 6 ng/µl of NdAg (triangle ) or the reaction buffer (open circle ) was added to the reaction mixture (as indicated by the arrow) for 10 min before the termination of trans-cleavage reaction. For the controls, PRP19S and KSR4 were pre-incubated with 0 ng/µl (bullet ) or 6 ng/µl of NdAg (black-square) at 37 °C for at least 20 min before the addition of 12 mM MgCl2. The fraction of uncleaved PRP19S was plotted against the trans-cleavage reaction time.

We then used the trans-cleavage reaction of PRP19S and KSR4 to study whether NdAg can be removed after it has facilitated the reconstitution of the hammerhead catalytic domain. NdAg (6 ng/µl) was extracted by phenol/chloroform after it has premixed with two RNAs (0.5 nM substrate and 5 nM ribozyme) for 10 min. Trans-cleavage reaction was then initiated by 12 mM MgCl2, and the cleavage of PRP19S was investigated. As shown in Fig. 4B, PRP19S was cleaved rapidly regardless of the removal of NdAg. Therefore, NdAg acts as an RNA chaperone. The phenol/chloroform extraction treatment appears to destabilize the PRP19S/KSR4 complex and decrease the extent of cleavage, and the addition of NdAg to the solution stimulated the cleavage of the remaining PRP19S (Fig. 4B).

Results of these studies regarding the cleavage of various substrates by different hammerhead ribozymes revealed that NdAg may act as an RNA chaperone that rapidly and efficiently facilitates the unfolding and refolding of RNA molecules range from <50 to ~900 nt in length. Consequently, the assembly of the hammerhead catalytic domain gets promoted by NdAg. Ribozyme RNAs may catalyze the cleavage of their cognate substrate RNAs once the trans-cleavage reactions are initiated by the addition of MgCl2.

Peptide NdAg Activates an Antisense RNA-- rKSS was designed to be a competitor of HH16. It is a 130-nt RNA that contains a copy of the complementary sequence of S near its 3' terminus. Nevertheless, the cleavage of S (0.5 nM) by HH16 (5 nM) was not perturbed by the presence of 0.5 to 5 nM of rKSS. Moreover, neither the premixing nor the co-denaturation and renaturation of rKSS and S prior to the initiation of trans-cleavage (by the addition of HH16 and MgCl2) affected the cleavage of S (data not shown). Therefore, rKSS appears to have stable intramolecular interaction that buries the complementary sequence of S.

We next investigated if peptide NdAg stimulates the antisense activity of rKSS. When 0.5 nM S was pre-mixed with 5 nM rKSS in the presence of NdAg for 30 min, the cleavage of S by 5 nM HH16 was inhibited. The pre-mixing period could be shorter (data not shown), and the inhibitory effect of rKSS was a function of NdAg concentration rather than a function of time (Fig. 5A). Moreover, with the facilitation of saturated concentrations of NdAg, 0.5 nM rKSS was sufficient to inhibit the cleavage of 0.5 nM S by 5 nM HH16, whereas an HDV cis-cleaving ribozyme did not have any inhibitory effect on the cleavage of S under the same condition (Fig. 5B). The near complete inhibition of S cleavage discloses that most of the S molecules are involved in the formation of S/rKSS hybrid. The substrate/competitor complex appears to be stable and prohibits further interaction of S and HH16. Therefore, NdAg facilitates rKSS becoming an antisense RNA.


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  		Fig. 5.   Effect of rKSS on the trans-cleavage reaction of S and HH16. A, 0.5 nM S and 5 nM rKSS were incubated with 0 (open circle ), 0.05 (bullet ), 0.1 (black-square), or 0.2 ng/µl NdAg (black-triangle) at 25 °C for 30 min prior to the initiation of trans-cleavage by the addition of 5 nM HH16 and 12 mM MgCl2. The cleavage of S by HH16 was plotted against the trans-cleavage reaction time. The data presented in dashed lines were from the corresponding control experiments that did not contain any rKSS. B, 0.5 nM S, 0.5 nM rKSS, and 0.5 ng/µl NdAg were premixed at 25 °C for 0 min (open circle ) or 30 min (bullet ) prior to the initiation of trans-cleavage by the addition of 5 nM HH16 and 12 mM MgCl2. As a negative control, 0.5 nM S, 0.5 nM HDV genomic RNA fragment 4-1, and 0.5 ng/µl NdAg were pre-mixed for 30 min (black-triangle) prior to the initiation of trans-cleavage. The cleavage of 0.5 nM S was plotted against the trans-cleavage reaction time. C, 0.5 nM rKSS was premixed with 0 (open circle ) or 0.5 ng/µl NdAg (black-triangle) at 25 °C for 30 min. The rKSS solution was then added to the reaction that contained 0.5 nM S, 5 nM HH16, and 12 mM MgCl2. The cleavage of 0.5 nM S was plotted against the trans-cleavage reaction time.

The action of NdAg was further characterized. rKSS did not have any inhibitory effect if the competitor RNA by itself was pre-mixed with NdAg (condition I) (Fig. 5C) or if NdAg was not mixed with the substrate and competitor RNAs for a certain period prior to the initiation of trans-cleavage (condition II) (Fig. 5B). In contrast, S was cleaved at an elevated rate under both conditions I and II because of the presence of NdAg in the reaction (Fig. 5C). It is likely that because of the longer size and lower concentration, the NdAg unfolded rKSS (130 nt, 0.5 nM) may not be able to compete with HH16 (38 nt, 5 nM) for S binding under condition I. Alternatively, when S is not close by, the NdAg unfolded rKSS may adopt some other structure(s) that prevents the binding of S (condition I). Although rKSS is shorter than most of the other RNAs used in this study, the unfolding of rKSS and the formation of rKSS and S hybrid appear to be relatively slow. As a result, only the stimulatory effect of NdAg on the reconstitution of hammerhead catalytic domain was observed under condition II.

Identification of the RNA Chaperone Domain of Hepatitis Delta Antigen-- To further narrow down the functional domain responsible for the catalytic stimulation of the hepatitis delta antigen, truncated versions of NdAg were made, and their effects on hammerhead ribozyme catalysis were examined. Peptides N5dAg and N7dAg are fusion proteins containing aa 14-59 and 24-75, respectively, of the hepatitis delta antigen. These peptides behaved analogously to peptide NdAg at 1) facilitating the cleavage of S (0.5 nM) by HH16 (5 nM), 2) promoting the trans-cleavage reaction of PRP19S (0.5 nM) and KSR4 (5 nM), and 3) stimulating the inhibitory effect of rKSS on the cleavage of S by HH16 (data not shown). The N5dAg and N7dAg concentrations required to achieve the maximum stimulatory effect on each reaction were 5- to 10-fold higher than that of NdAg (data not shown). Therefore, the core of the RNA chaperone domain appears to reside in aa 24-59 of the hepatitis delta antigen, which interacts with non-HDV RNA. Neither the arginine-rich sequence near the N terminus nor the arginine-rich motif of the middle one-third of the hepatitis delta antigen that has been shown to bind HDV RNA specifically (8-10) is required for RNA chaperone activity. The N-terminal arginine-rich sequence nevertheless may increase the nucleic acid binding affinity of hepatitis delta antigen peptides. As a result, NdAg facilitates hammerhead ribozyme catalysis and RNA unfolding and refolding at lower concentrations than those of N5dAg and N7dAg.

The RNA chaperone domain of the hepatitis delta antigen is rich in basic amino acids, especially lysine (Fig. 1A). We next examined if the highly basic peptide KKKKKKK (K7) mimics the effect of hepatitis delta antigen peptides in facilitating the single turnover reaction of S and HH16. The results indicated that peptide K7 did not accelerate the rate of cleavage unless its concentration was >5,000-fold higher than that of N5dAg, N7dAg, or NdAg (data not shown). Therefore, similar to NCp7 and A1 CTD (24), some features in addition to the positively charged groups of the RNA chaperone domain of the hepatitis delta antigen peptides are important for the promotion of hammerhead ribozyme catalysis. Because a peptide containing the RNA chaperone domain of the hepatitis delta antigen is responsible for the coiled-coil multimer formation (28, 29), it is likely that in addition to the high density positively charged groups, the alpha -helical structure and/or the formation of a peptide multimer are important for RNA chaperone activity. The structural and functional relationship of the RNA chaperone domain of the hepatitis delta antigen requires further elucidation.

Hepatitis Delta Antigen Affects the Folding of HDV RNA-- Each sense of HDV RNA contains an autolytic domain that may fold into a pseudoknot-like structure and then undergoes site-specific cis-cleavage (15, 16). Previous studies illustrate that presumably because of the highly self-complementary nature of HDV RNA, the sequence surrounding the autolytic domain may cause the formation of alternative structures that interfere or prevent the adoption of the autocatalytic structure. Consequently, the cis-cleaving activities of different HDV RNA subfragments may vary significantly although each of them contains an intact autolytic domain (30). This characteristic of HDV RNA was evident by the HDV genomic RNA fragments synthesized in this study (Fig. 6, A and B): 4-1 (681-775; i.e. nt 681 to nt 775 of HDV genomic RNA), as well as 1-2 (583-800) that underwent cis-cleavage when the reactions were carried out in the presence of 12 mM MgCl2 at 37 °C. The rate of cleavage of the former was significantly higher than that of the latter; 2-2 (625-800) cis-cleaved slowly and inefficiently under the same condition; and 3-2 (659-800) together with 2-4 (625-860) barely cis-cleaved. However, the cis-cleaving activities of 2-2, 2-4, and 3-2 can be elevated significantly if the RNA molecules have gone through repeated cycles of heat denaturation and renaturation (30) prior to the initiation of cis-cleavage, or if the cis-cleavage reaction is performed in the presence of moderate concentrations of denaturant (at 37 °C) or at higher temperatures (50 °C) (data not shown).


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  		Fig. 6.   Effect of NdAg on the cis-cleavage reaction of HDV genomic RNA fragments. A, schematic diagram of different RNA fragments. The gray box represents the autolytic domain of HDV RNA of genomic sense, and the arrowhead indicates the cleavage site. The nomenclature of the nucleotide of HDV RNA is according to Makino et al. (1987). B, RNA fragments were incubated with 0 (open circle ) or 10 ng/µl of NdAg (bullet ) at 37 °C for 30 min after they were heat-denatured and -renatured. The cis-cleavage reaction of each RNA fragment was initiated by the addition of 12 mM MgCl2, and the fraction of uncleaved precursor RNA was plotted against the cis-cleavage reaction time.

To investigate whether the RNA chaperone activity of hepatitis delta antigen peptides identified through the studies of hammerhead ribozyme catalysis is biologically relevant, we studied the effect of peptide NdAg on the cis-cleaving activities of the HDV genomic RNA fragments described above. As shown in Fig. 6B, the premixing with NdAg prior to the initiation of cis-cleavage altered the ribozyme activity of three fragments: the cis-cleaving activity of 4-1 was abolished; 3-2 became active although it cis-cleaved slowly; and a large portion of the inactive molecules of 2-2 were activated and consequently the extent of cleavage was elevated from <50% to >=  90%. Preincubation with NdAg did not have detectable effect on 2-4 or 1-2; 2-4 remained inactive and neither the rate nor the extent of cis-cleavage of 1-2 were altered (Fig. 6B). These results disclose that NdAg may modulate the autocatalytic activity of HDV RNA subfragments by stimulating RNA unfolding and refolding.

We next examined the activity of other hepatitis delta antigen peptides on the cis-cleavage of fragment 4-1, which is the smallest RNA fragment containing the autolytic domain of HDV genomic RNA. The results illustrate that similar to that of NdAg, peptides dAg, NMdAg, N5dAg, and N7dAg attenuated the ribozyme activity of 4-1 (data not shown). Peptide CdAg, in contrast, did not have a detectable effect on the cis-cleavage reaction of 4-1 (data not shown). This finding discloses that the functional domain resides in aa 24-59 of hepatitis delta antigen. In addition, neither the arginine-rich sequence nor the arginine-rich motif of hepatitis delta antigen that have been shown to bind HDV RNA specifically (8-13) are required for the activity identified herein.

In summary, we have shown that the RNA chaperone domain of the hepatitis delta antigen can modulate the autocatalytic activity of HDV RNA in vitro. Conceivably, the modulation of the ribozyme activity of HDV RNA by hepatitis delta antigen is one of the mechanisms that regulates HDV replication.

    ACKNOWLEDGEMENTS

We thank Huey-Wen Huang and Sing-Yen You for assistance in protein purification and D. Platt for English editing.

    FOOTNOTES

* This work was supported by Academia Sinica, Republic of China, and a grant from National Science Council, Republic of China (NSC-86-2311-B-001-045-B21).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 886-2-2-7883134; Fax: 886-2-2-7826085; E-mail: hnwu{at}gate.sinica.edu.tw.

The abbreviations used are: HDV, hepatitis delta virus; nt, nucleotide(s); aa, amino acid(s); SdAg, small delta antigen; LdAg, large delta antigen; CTD, C-terminal domain.
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Abstract
Introduction
Procedures
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References

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