A cell-based reporter assay for inhibitor screening of hepatitis C virus RNA-dependent RNA polymerase.
The hepatitis C virus (HCV) NS5B, a RNA-dependent RNA polymerase (RdRp), is an attractive target for anti-HCV agents. The major disadvantages of the commonly used polymerase inhibitor screening involving the assessment of in vitro RNA synthesis are that it is incapable of demonstrating the cellular permeability and the cytotoxicity of compounds. To overcome these limitations, we created the BHK-NS5B-FRLuc reporter cell line that carries stably transfected NS5B and a bicistronic reporter gene, (+)FLuc-(-)UTR-RLuc, which can be used to simultaneously measure cellular toxicity and intracellular RdRp activity. The (+)FLuc-(-)UTR-RLuc construct comprises the firefly luciferase (FLuc) gene and the Renilla luciferase (RLuc) gene in reverse orientation flanked by both negative strands of the HCV 5'- and 3'-untranslated regions (UTRs), in which FLuc and RLuc reporter proteins are regulated by host polymerase and functional NS5B polymerase, respectively. The reporter system was validated with specific agents against NS5B polymerization. Additionally, this assay was placed in 96-well plates and had a Z'-factor value of approximately 0.75, which is amenable for facilitating high-throughput screening operations. Notably, in combination with the structured-based virtual screening, an imidazole derivative compound was evaluated as a candidate HCV RdRp inhibitor.
Lee
Jin-Ching
JC
Department of Biotechnology, College of Life Science, Kaohsiung Medical University, San Ming District, Kaohsiung City, Taiwan, ROC. jclee@kmu.edu.tw
Tseng
Chin-kai
CK
Chen
Kuan-Jen
KJ
Huang
Kuo-Jung
KJ
Lin
Chun-Kuang
CK
Lin
Ying-Ting
YT
D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D017930:Genes, Reporter; D016174:Hepacivirus / Q000201:enzymology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D019380:Anti-HIV Agents; D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase