A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HD V contain a ribozyme that undergoes cis-cleavage, thereby processing the tr anscript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have att enuated self-cleavage. Structure mapping and site-directed mutagenesis reve aled an inhibitory stretch consisting of upstream nucleotides -24 to -15 th at forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3 ')) located at the very 3'-end of the ribozyme. Two other alternative pairi ngs were found, Alt 2, which involves upstream nucleotide-ribozyme interact ions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleava ge was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased t o 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stret ch in cis. This hairpin is followed by a stretch of single-stranded pyrimid ine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, a nd that the J(-1/1) stretch is conserved among antigenomic HDV isolates. La stly, the secondary structure of the Alt 1-containing ribozyme provides ins ight into possible folding intermediates of the ribozyme. (C) 2000 Academic Press.