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Dual luciferase assay to assess the replication of the hepatitis C virus subgenomic replicon

Sarah Gretton, Mark Harris Faculty of Biologial Sciences, University of Leeds 09/2008

Hepatitis C virus (HCV) is a global health problem. Currently, there is no vaccine for HCV and therapy is only effective in around 50 % of individuals. Therefore, improved understanding of the viral life cycle and systems for screening viral inhibitors are required.

 

The development of a subgenomic replicon system, encoding only the viral non-structural proteins required for RNA replication, allows the study of replication of the virus, without the release of infectious particles and the necessary containment facilities required for this work.

 

Here, we directly assess the effects of potential inhibitors on both the RNA replication of HCV, and overall translation in hepatoma cells using a BMG LABTECH microplate reader. A subgenomic replicon has been generated from the efficiently replicating genotype 2a strain of HCV (JFH1), incorporating the firefly luciferase gene under the control of the HCV internal ribosome entry site element. As an indicator for overall translation in the cell, a capped RNA transcript from the pRLTK plasmid was used.

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