Z Gastroenterol 2005; 43 - P272
DOI: 10.1055/s-2005-920052

Inhibition of Hepatitis C Virus Subgenomic Replication Cycle by Constitutive Transport Element Linked Ribozymes Targeting Highly Conserved 5' and 3' Untranslated Regions

D Jarczak 1, M Korf 1, MP Manns 1, M Krüger 1
  • 1Abteilung für Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover, Hannover

Background/Aims: The infection with Hepatitis C Virus (HCV) can lead to the development of chronic liver disease with limited therapeutic options in a significant proportion of patients. Hairpin ribozymes (Rz) are small RNA molecules with endonuclease activity that can be engineered to specifically cleave RNA sequences. However, activity depends on Rz expression, target colocalization and cleavage site accessibility. CTE, an RNA motif has previously shown to interact with intracellular RNA helicases and might enhance cleavage activity by enhancement of RNA binding and unwinding activities of RNA helicase. CTE has also been shown to facilitate nuclear export, thereby potentially increasing cytoplasmatic Rz abundance and colocalization with HCV-RNA substrate. The aim of our study was to investigate cleavage activities of endogenously expressed CTE-linked Rz on 5'- and 3'-UTR HCV RNA.

Methods: Retroviral vectors expressing Rz or Rz-CTE from a tRNA- or U6-promoter were transfected into Huh7-cells i)with the luciferase reporter construct pRL–5'FL–3'UTR (Renilla-HCV-IRES-Firefly–3'UTR) and analyzed for the luciferase activity. ii) stably expressing a monocistronic subgenomic HCV replicon and analyzed in western and northern blot experiments.

Results: Transfected cells demonstrated high ribozyme expression levels (ca. 60 000 copies/cell) and significant reduction up to 40% in HCV replication levels. However, attachment of CTE to the ribozyme 3'-end did not significantly alter ribozyme cleavage activity in subgenomic HCV replicon cells.

Conclusions: From these observations we conclude that ribozymes significantly reduce HCV RNA expression in subgenomic HCV replicon cells but the attachment of CTE to ribozymes does not significantly enhance anti-HCV activity of selected ribozymes.

Keywords: RNA interference, constitutive transport element, hepatitis C virus, ribozyme