The pentapurine sequence GGAGA, located between 80 and 84 nucleotides downstream of the cleavage site in the self-cleaving antigenomic RNA of hepatitis delta virus, is necessary for highly efficient cleavage and for stability in up to 20 M formamide. Yet much of the cleavage activity lost upon its removal from the 3' end of an 84-nucleotide RNA can be restored by elongation of the 5' end of the RNA with the polypyrimidine sequence found in the virus. We propose that this sequence alteration causes a refolding of the RNA, resulting in a "structural compensation" of the active core of the molecule. Restoration of the self-cleavage activity did not restore the ability to cleave in high concentrations of formamide. Deletion mutagenesis was carried out and supported the alternate RNA folding. The ability to assume more than one active conformation and for one RNA structure to compensate for another in supporting ribozyme activity may be unique to RNA enzymes and could be a useful adaptation in viruses or in prebiotic RNAs.

RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.

The DNA of a nuclear polyhedrosis virus (NPV) propagated in the larvae of S. litura was analysed with AluI, ApaI, BamHI, BglII, BstEII, BstNI, ClaI, EcoRI, EcoRV, HindIII, HinfI, KpnI, MspI, PstI, PvuII, SalI, Sau3AI, SmaI, TaqI, XbaI, and XhoI. The unique restriction endonuclease profiles of the virus indicate it to be a distinct NPV isolate. The size of the viral genome was estimated to 132 kbp; the genome was mapped with HindIII and PstI.

Self-splicing and self-cleaving RNAs have been engineered to function as antisense RNAs by defining separate enzyme and substrate moieties.'-' Although these RNAs are efficient in cleaving the substrate RNAs in vitro, none has yet been shown to work enzymatically in vivo. The hepatitis delta RNAs offer higher efficiency of cleavage and stability than do any other ribozymes.Hepatitis delta virus is a unique mammalian virus, a single-stranded, circular RNA of only 1679 nucleotides (nts). We detected self-cleavage activity of longer-thanunit-length genomic and antigenomic R N A S .~.~ Subsequently, both RNAs were truncated to 86 genomic nts6 and 89 antigenomic nts, without loss of activity. In fact, the antigenomic RNA cleaved more efficiently at 37°C than has any other selfcleaving RNA yet reported, cleaving to completion in less than 15 seconds.'Although the computer-derived structure of the genomic RNA generated a central core in RNAs ranging from 154 to 86 nts (FIG. 1A),8 the structure of the antigenomic RNA, even in the same size class, differed greatly. As the RNAs are 70% similar in sequence, however, we generated an analogous antigenomic structure by analysis of possible conserved base pairs (FIG. 1B). A striking feature in both molecules was the extended stem 4 structure. To determine if this stem region was required for catalytic activity, deletions were introduced into the cloned cDNA. The resulting RNAs transcribed in vitro9 were assayed for self-cleavage at 37°C in 50 mM Tris buffer, p H 7.5, and 10 mM MgClz for 5 minutes.When the self-cleavage of the internally truncated genomic RNA G73 was assayed (TABLE 1) and compared with the extended stem 4 parent RNA G86 in the absence of formamide, cleavage was reduced sixfold, from 59 to 9%. To test the effect of formamide on the cleavage reaction, first the RNAs were incubated in the presence of formamide at 37°C for 5 minutes, and then 10 mM magnesium was added, followed by an additional 5 minutes of incubation. The formamide enhanced the cleavage of (386, and complete cleavage was seen in 5 minutes. This presumably was due to the denaturing of inactive RNA conformers, allowing formation of the active RNA and self-cleavage. The truncated G73 completely lost activity in the presence of formamide, and no cleavage was seen at all. Rather than allow structure to form, the formamide appeared to denature the active site of the molecule, preventing cleavage. Thus, an extended stem 4 is essential to the active core of the genomic RNA in the presence of formamide.The antigenomic RNA had previously been noted to be unusually resistant to formamide denaturation even in comparison with the genomic RNA.7J0,L' We identified a sequence element, GGAGA, at the 3' terminus of the 89 nucleotide 277

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