Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system
Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system
复制标题
利用细胞分泌型碱性磷酸酶报告复制子系统筛选丙型肝炎病毒的抗病毒活性
DOI：
10.1016/j.antiviral.2005.03.006
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发表时间：
2005-08-01
影响因子：
7.6
通讯作者：
Lemon, SM
中科院分区：
文献类型：
--
作者：
Bourne, N;Pyles, RB;Lemon, SM
We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV. (c) 2005 Elsevier B.V. All rights reserved.
