Hepatitis C virus (HCV) NS5B, an RNA-dependent RNA polymerase (RdRp), is an attractive target for antiviral agents. The in vitro RNA synthesis system based on radioisotopic readout is commonly used for polymerase inhibitor screening; however, this system generates large amounts of radioactive waste and is not amenable to high-throughput applications. To overcome this limitation, we generated pFLuc-(-)UTRΔC-RLuc, a bicistronic reporter vector, which allows effective and sensitive distinction of RdRp activity by using a cell-free coupled transcription/translation system. This reporter construct comprises the firefly luciferase (FLuc) and the Renilla luciferase (RLuc) genes in reverse orientation flanked by the two negative strands of the HCV 5'- and 3'-untranslated regions in which FLuc and RLuc reporter proteins are regulated by bacteriophage T7 polymerase and NS5B polymerase, respectively. The increase in RLuc activity was proportional to the amount of active RdRp. This cell-free dual reporter system was further validated using specific RdRp inhibitors. Hence, linear dose-response curves between RLuc activity and specific inhibitors were obtained, as was faster drug screening through real-time measurement of chemiluminescence. Moreover, this reporter system is suitable for robust in vitro screening because of a statistically acceptable Z' factor value of 0.79 under the antiviral screening condition in the 96-well format.
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