A 3-DIMENSIONAL MODEL OF HEPATITIS-DELTA VIRUS RIBOZYME BASED ON BIOCHEMICAL AND MUTATIONAL ANALYSES

Background: Hepatitis delta virus (HDV), which has a single-stranded R NA genome about 1700 nucleotides long, is a satellite virus of hepatit is B, and is associated with a high incidence of fulminant hepatitis a nd death in infected humans. Like certain pathogenic subviral RNAs tha t infect plants, HDV RNA features a closed-circular conformation, a ro lling-circle mechanism of replication and RNA-catalyzed self-cleaving reactions of both genomic and anti-genomic strands in vitro. The catal ytic domains cannot be folded into either the hammerhead or hairpin se condary-structure motifs that have been found in other self-cleaving R NAs. Results: A pseudoknot secondary-structure model has been suggeste d for the catalytic domain (ribozyme) of HDV RNA. We conducted extensi ve mutational analyses of regions of the HDV ribozyme predicted in thi s model to be single stranded, and found that several of them are impo rtant for catalytic activity. We used these data, sequence comparisons between different isolates and previously published structural analys es to produce a computer graphic model of the three-dimensional archit ecture of the HDV ribozyme. Conclusions: Our model supports the pseudo knotted structure and rationalizes several observations relating to th e lengths of the various stems and the sequence requirements of the si ngle-stranded regions. It also provides insight into the catalytic mec hanism of the HDV ribozyme. We specifically propose that residues C75, U20 and C21 form the basis of the catalytic region and are close to t he cleavable phosphate.