Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons

TY - JOUR

T1 - Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons

AU - Jarczak, Dominik

AU - Korf, Mortimer

AU - Beger, Carmela

AU - Manns, Michael P

AU - Krüger, Martin

PY - 2005/11

Y1 - 2005/11

N2 - Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5'- and 3'-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5'- and 3'-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNA(Val), U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3'/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5'- or 3'-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3'-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNA(Val) as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3'-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNA(Val)-driven Rzs and U6-derived siRNAs both directed against highly conserved 5'- and 3'-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.

AB - Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5'- and 3'-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5'- and 3'-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNA(Val), U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3'/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5'- or 3'-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3'-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNA(Val) as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3'-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNA(Val)-driven Rzs and U6-derived siRNAs both directed against highly conserved 5'- and 3'-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.

KW - Blotting, Western

KW - Carcinoma, Hepatocellular

KW - Cell Line, Tumor

KW - Conserved Sequence

KW - Genes, Reporter

KW - Genes, Viral

KW - Genetic Vectors

KW - Genome, Viral

KW - Hepacivirus

KW - Humans

KW - Liver Neoplasms

KW - Luciferases

KW - Models, Biological

KW - Protein Biosynthesis

KW - RNA, Catalytic

KW - RNA, Small Interfering

KW - RNA, Viral

KW - Replicon

KW - Virus Replication

U2 - 10.1111/j.1742-4658.2005.04986.x

DO - 10.1111/j.1742-4658.2005.04986.x

M3 - SCORING: Journal article

C2 - 16279954

VL - 272

SP - 5910

EP - 5922

JO - FEBS J

JF - FEBS J

SN - 1742-464X

IS - 22

ER -