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 no t been previously identified in the HDV ribozymes. P1.1 consists of two can onical 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 antig enomic ribozyme. In this study, the base pairing requirements of P1.1 for c leavage activity were tested in both the genomic and antigenomic HDV ribozy mes by mutagenesis. In both sequences, cleavage activity was severely reduc ed when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential: s tructural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structu re should also be modified to include P1.1.