Examination of the folding pathway of the antigenomic hepatitis delta virus ribozyme reveals key interactions of the L3 loop

With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5'-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme-substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the "on" folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.     

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA.

To determine the sequence requirements and structural features of the self-cleavage domain of hepatitis delta virus (HDV) antigenomic RNA, we constructed a series of mutants and measured the rate constant of the cleavage reaction for each. The self-cleavage activity of HDV RNA of antigenomic sense was found to reside in a region of less than 90 nucleotides in length. The catalytic domain contained a long complementary sequence which could be deleted to half of its original size. Moreover, this region could be replaced by other sequences as long as they could fold into a stem-and-loop structure. The catalytic domain also required a 6-basepair helix adjacent to the cleaving point for activity. The structural features of these two base-pairing regions are quite similar to those of the HDV genomic self-cleavage domain. The cleavage site as well as the the hinge region (the sequence between the two stems) requires specific sequences for activity.     

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.     

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme.

We conducted extensive mutagenesis analysis on a hepatitis delta virus (HDV) genomic ribozyme to study the sequence specificity of certain region and to derive the secondary structure associated with the catalytic core. The results confirmed that the autocatalytic domain of HDV genomic RNA contained four base-pairing regions as predicted in the 'pseudo-knot' model [Perrotta & Been (1990) Nature 350, 434-436]. The size and sequence of one of the base-pairing regions, i. e. stem-and-loop, could be flexible. Helix 3 and the first basepair of helix 1 required specific sequence to retain self-cleavage activity. The structural requirement of helix 2 was less stringent than the other base-pairing regions. Moreover, the size of helix 1 affected self-cleavage whereas the length of hinge could be variable even though the first three residues of hinge had stringent sequence requirement.     

A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide.

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 degrees C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a polypurine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82 to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1 of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1 severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions.     

Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme.

Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested by site-directed mutagenesis. Two models in which bases 5' to the cleavage site are paired with sequence at the 3' end of the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence was consistent with a third model in which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage in trans with a 15-mer ribozyme, thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.     

cDNA sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus.

Callitrichid hepatitis is an infection of New World primates caused by an arenavirus, currently referred to as callitrichid hepatitis virus, that is closely related to lymphocytic choriomeningitis virus (LCMV). We have cloned and sequenced the GP-C gene of callitrichid hepatitis virus and found that the cDNA sequence is 84 to 86% identical to those of the GP-C genes of LCMV strains Armstrong and WE, while the deduced amino acid sequence is 95 to 96% identical to those of the GP-C gene products of the same strains. This high degree of similarity indicates that the etiologic agent of callitrichid hepatitis is in fact LCMV. The wide geographic distribution of callitrichid hepatitis outbreaks in the United States serves as a reminder that LCMV is also a human pathogen whose public health implications are not well understood.     

Partial sequence analysis of 130 randomly selected maize cDNA clones.

As part of a project to identify novel maize (Zea mays L. cv B73) genes functionally, we have partially sequenced 130 randomly selected clones from a maize leaf cDNA library. Data base comparisons revealed seven previously sequenced maize cDNAs and 18 cDNAs with sequence similarity to related maize genes or to genes from other organisms. One hundred five cDNAs show little or no similarity to previously sequenced genes. Our results also establish the suitability of this library for large-scale sequencing in terms of its large insert size, proper insert orientation, and low duplication rate.     

Inhibition of the catalytic activity of trans-acting antigenomic delta ribozyme by selected antibiotics and their Cu2+ complexes

The effect of selected 10 antibiotics and their complexes with Cu(2+) ions on the catalytic activity of the trans-acting antigenomic delta ribozyme was investigated. Sisomicin, vancomycin, and actinomycin D displayed weak inhibitory properties. However, much stronger effects were detected with complexes of these antibiotics with Cu(2+) ions. The strongest inhibition was observed with actinomycin D-Cu(2+) complex, for which the calculated K(i) value was reduced ca. 35-fold upon metal ion complexation. We postulate that the antibiotic-Cu(2+) complexes are guided to the ribozyme metal ion binding site(s) presumably displacing the catalytically important metal ion(s). Moreover, we assume that, once positioned in appropriate distances to RNA phosphate groups and bases, the coordinated Cu(2+) ions become positively charged factors that enhance the affinity of the antibiotics to the ribozyme. These observations indicate that coordination of metal ions to antibiotics substantially changes their properties which might also have a biological relevance inside the cell.     

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.     

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2′,3′-cyclic phosphate and 5′-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson–Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme–substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

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

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.     

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.     

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.     

Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.

The cleavage reaction catalyzed by the trans -acting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed with the SR13(Rp) substrate was, at most, 20% of this value (with 0.5-100 mM Mg2+ions) at pH 5.0. Nonetheless, the value of kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.