Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications

Abstract

cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

“…The switch from small to large antigen is the result of a mutation that eliminates the amber stop codon (codon 196) that defines the terminus of the small antigen, such that transcription results in a message that is translated to a form of the antigen that has an additional 19 amino acids (Casey and Gerin, 1995). Although neither form of the protein has been shown to have enzymatic activity, the RNA of the virus does code for an enzyme, an RNA enzyme, or ribozyme (Kuo et al, 1988b;Sharmeen et al, 1988;Wu et al, 1989). The self-cleaving activity of this ribozyme appears to be required for replication of the viral RNA (Jeng et al, 1996a;Macnaughton et al, 1993), which supports a hypothesized role for the ribozyme in processing replication products (Kuo et al, 1988b;Sharmeen et al, 1988;Wu et al, 1989).…”

Restoration in vivo of defective hepatitis delta virus RNA genomes

“…We noted that in lanes 6 and 7 of Figure 2B there was an indication of a minor heterogeneous species migrating faster, but there were not any discrete-sized species that migrated as for 1679-nt linears or circles. Thus we interpreted that the heterogeneous species detected 2 copies of the HDV genome and two copies of the genomic ribozyme (shaded circles) was modified at either of two unique locations, the XbaI site at position 785/786 or the NheI site at position 430/431, using a published numbering system (Kuo et al 1988a). If these DNA were transfected into cells, we would expect a primary transcript of genomic RNA, containing two ribozymes, to be processed by cleavage followed by ligation and produce unit-length RNA circles.…”

“…Originally, constructs expressing mutated HDV genomes were based on vector pDL553, which expresses a 1.23 of wild-type HDV genomic RNA . As represented in Figure 1, non-HDV sequences were inserted at one of two unique locations: XbaI site (position 785/786) or NheI site (position 430/431), using the numbering of a published HDV RNA sequence (Kuo et al 1988a). The insertions are summarized in Table 1.…”

“…Plasmids were selected and subjected to automated nucleotide sequencing. Resulting sequences were analyzed relative to the HDV master sequence (Kuo et al 1988a) using the program Sequencher 4.2.1 (Gene Codes).…”

“…However, a major contributor to this redirected transcription is considered to be the structure of the genomic and antigenomic RNAs. These circular RNAs have the ability to fold, by intramolecular basepairing involving z74% of all nucleotides, to produce the unbranched rodlike structure (Wang et al 1986;Kuo et al 1988a). There is, in addition, evidence that the dAg has not only RNA-binding ability but also the potential to make specific interactions with such rodlike HDV RNAs (Chao et al 1991;Ryu et al 1993).…”