期刊
INTERVIROLOGY
卷 48, 期 5, 页码 301-311出版社
KARGER
DOI: 10.1159/000085099
关键词
hepatitis C virus; subgenomic replicon; interferon-alpha; cyclosporin A; interferon-stimulated response element; firefly luciferase; beta-galactosidase
类别
资金
- NATIONAL CANCER INSTITUTE [R01CA089121] Funding Source: NIH RePORTER
- NCI NIH HHS [CA89121] Funding Source: Medline
Interferon-alpha (IFN alpha) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways. are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFN alpha was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFN alpha. These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV. Copyright (C) 2005 S. Karger AG, Basel.
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