Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation

Ribozymes are small catalytic RNA molecules that can be engineered to enzym atically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With the ir simple structures and site-specific cleavage activity, they have been ex ploited as potential therapeutic agents in a variety of human disorders, in cluding hepatitis C virus (HCV) infection. We have designed a hairpin riboz yme (Rz3 'X) targeting the HCV minus-strand replication intermediate at pos ition 40 within the 3 'X tail. Surprisingly, Rz3 'X was found to induce gan ciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES) -dependent translation of her pes simplex virus thymidine kinase (TK). Rz3 'X-transduced GCV-resistant He La reporter cells showed substantially reduced IRES-mediated HCV core prote in translation compared with control vector-transduced cells. Since these r eporter systems do not contain the HCV 3 'X tail sequences, the results ind icate that Rz3 'X probably exerted an inhibitory effect on HCV IRES activit y fortuitously through another gene target. A novel technique of ribozyme c leavage-based target gene identification (cleavage-specific amplification o f cDNA ends) (M. Kruger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-S taal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome a -subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3 'X. We t hen showed that additional ribozymes directed against PSMA7 RNA inhibited H CV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK r eporter cell system and a transient transfection assay performed with a bic istronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In con trast, ribozymes were inactive against IRES of encephalomyocarditis virus a nd human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dos e-dependent inhibitory effect on HCV IRES-mediated translation but not on c ap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.