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. (+info)
Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles.
The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196. (+info)
Cell cycle arrest mediated by hepatitis delta antigen.
Hepatitis delta antigen (HDAg) is the only viral-encoded protein of the hepatitis delta virus (HDV). This protein has been extensively characterized with respect to its biochemical and functional properties. However, the molecular mechanism responsible for persistent HDV infection is not yet clear. Previously, we reported that overexpression of HDAg protects insect cells from baculovirus-induced cytolysis [Hwang, S.B. Park, K.-J. and Kim, Y.S. (1998) Biochem. Biophys. Res. Commun. 244, 652-658]. Here we report that HDAg mediates cell cycle arrest when overexpressed in recombinant baculovirus-infected insect cells. Flow cytometry analysis has shown that HDAg expression in Spodoptera frugiperda cells causes an accumulation of substantial amounts of polyploid DNA in the absence of cell division. This phenomenon may be partly responsible for the persistent infection of chronic HDV patients. (+info)
A nested double pseudoknot is required for self-cleavage activity of both the genomic and antigenomic hepatitis delta virus ribozymes.
The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1. (+info)
Characterization of the 5' ends for polyadenylated RNAs synthesized during the replication of hepatitis delta virus.
The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5' rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5' ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5' ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5' ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5' ends. The observed end located at nt 1630, and most of the novel 5' ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5' ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5' ends could be controlled by nucleotide sequences at and around nt 1630. (+info)
Mimicry of the hepatitis delta virus replication cycle mediated by synthetic circular oligodeoxynucleotides.
BACKGROUND: Hepatitis delta virus (HDV) is a circular single-stranded RNA pathogen whose monomeric form results from self-processing. Although studies have examined minimal HDV ribozyme activities, the mechanism for forming the circular virus remains unclear, and the trans catalytic properties of self-processed forms of HDV ribozymes have not been studied. In addition, HDV ribozymes have not previously been engineered to cleave a non-HDV sequence. RESULTS: Long repeating RNAs have been produced from in vitro rolling-circle transcription of synthetic circular oligodeoxynucleotides encoding catalytically active subsets of the entire antigenomic RNA virus. Like full-length HDV, these multimeric RNAs undergo self-processing to monomer length; importantly, cyclization is found to occur efficiently, but only in the presence of the circular template. Linear and circular monomer ribozymes and engineered variants are shown to be active in cleaving HDV and HIV RNA targets in trans, despite having self-binding domains. CONCLUSIONS: Mimicry of the rolling-circle replication pathway for HDV replication has led to a new proposal for cyclization of HDV RNA. Under these conditions, cyclization is mediated by the complementary circular template. In addition, it has been shown that self-processed HDV ribozymes can be catalytically active in trans despite the presence of antisense sequences built into their structure. (+info)
Unique properties of the large antigen of hepatitis delta virus.
The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [(14)C]mevalonolactone and is interpreted to be isoprenylated L (L(i)). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by (14)C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, L(i) was preferentially assembled into virus-like particles. (ii) L(i) could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly. (+info)
Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.
We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme. (+info)