Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Abstract

Hepatitis delta virus (HDV) is a small single‐straded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self‐cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The self‐cleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling‐circle replication to unit‐length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysis has not yet been proven. A minimal natural ribozyme sequence with proficient in vitro self‐cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (P1–P4). The two pairings that define the 5′ and 3′ boundaries of the ribozyme, P1 and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo‐cross‐linking, mutagenesis and computer‐assisted modeling provides clues to the three‐dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5′ end of P1, is in close proximity to three single‐stranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans‐acting ribozymes and smaller more active cis‐acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.