RNase footprinting demonstrates antigenomic hepatitis delta virus ribozyme structural rearrangement as a result of self-cleavage reaction.
BACKGROUND
Hepatitis delta virus (HDV) is a satellite virus of hepatitis B. During viral replication the 1700-nucleotide-long genomic RNA and its complement, the antigenomic RNA, undergo self-cleavage catalyzed by internal ribozyme motifs that are essential for propagation of the virus in vivo. These self-cleavage activities are provided by 85-nucleotide-long sequence elements, the genomic and antigenomic forms of HDV ribozyme. Recently four permuted variants of the antigenomic HDV cis-ribozyme with a self-cleavage site located at the 5' proximity, in the middle, or nearby the 3' end of the molecule were constructed and synthesized. These constructs exhibit equal activity, a bi-phasic kinetics of self-cleavage reaction and reaction products with low and high stability. We have used ribonuclease probing to footprint the structures of uncleaved and post-cleaved forms of the antigenomic HDV ribozymes in solution. Uncleaved ribozymes, associated and individual products of the self-cleavage reaction were analyzed using ribonuclease and Fe(II)-EDTA protection assays to reveal the differences in the structure of pre- and post-cleaved antigenomic HDV ribozyme in solution.


RESULTS
Our findings demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, in contrast to minor conformational switch observed in crystals of the genomic form. This study indicates that changes in the structure of stem P1 and stem P4 are minor, those of the region ascribed to stem P2, stem P3 and loop l3 are dramatic, while stem P1.1 results from the self-cleavage reaction.


CONCLUSIONS
Our data agree with the structure of post-cleaved and disagree with that of pre-cleaved forms of HDV ribozyme published elsewhere.
Savochkina
Larissa
L
Russian Cardiology Research and Production Center, 121552 Moscow, Russia. lasav@cardio.ru
Alekseenkova
Victoria
V
Belyanko
Tatyana
T
Dobrynina
Nadezhda
N
Beabealashvilli
Robert
R