Cleavage of viral RNA and inhibition of viral translation by hepatitis C virus RNA-specific hammerhead ribozyme in vitro.
OBJECTIVE
A hammerhead ribozyme has been used as a new way to suppress specific gene expression. We designed hammerhead ribozymes directed against hepatitis C virus RNA, and investigated their cleavage efficiency and inhibitory effect on viral translation in vitro.


METHODS
Three hammerhead ribozymes bearing different cleavage sites in the core region of hepatitis C virus RNA (genotype 1b) were designed in this study. Ribozymes and the target hepatitis C virus RNA were synthesized by in vitro transcription. The cleavage efficiency was evaluated by the ribozyme cleavage assay. The inhibitory effect of the ribozyme on viral translation was further studied by the viral translation inhibition assay.


RESULTS
All ribozymes specifically cleaved the target RNA of 1217 bases at a physiological temperature in a dose-dependent manner, with the specific cleavage increasing with a longer incubation period. The target RNA was cleaved most efficiently by the ribozyme with the cleavage site located nearest to the initiation codon. In the viral translation inhibition assay, all ribozymes showed a significant inhibitory effect on viral translation. The ribozyme with the cleavage site located farthest from the initiation codon blocked viral translation most efficiently, and demonstrated almost 70 to 80% inhibition. For ribozymes with the T7 transcription terminator sequence, both the target RNA cleavage and the inhibition of viral translation tended to be achieved less efficiently by ribozymes with T7 terminator than by those without it.


CONCLUSIONS
These findings suggest that ribozyme-mediated hepatitis C virus RNA cleavage may serve as a new strategy in the treatment of hepatitis C virus infection.
Ohkawa
K
K
First Department of Medicine, Osaka University School of Medicine, Suita, Japan.
Yuki
N
N
Kanazawa
Y
Y
Ueda
K
K
Mita
E
E
Sasaki
Y
Y
Kasahara
A
A
Hayashi
N
N
D016174:Hepacivirus / Q000235:genetics*; D007700:Kinetics; D010443:Peptide Chain Termination, Translational; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*
D016337:RNA, Catalytic; D012367:RNA, Viral