Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model

To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), whic...

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Veröffentlicht in:World journal of gastroenterology : WJG 2010-11, Vol.16 (44), p.5582-5587
Hauptverfasser: Fu, Qiu-Xia, Wang, Li-Cui, Jia, Shuai-Zheng, Gao, Bo, Zhou, Yong, Du, Juan, Wang, Ying-Li, Wang, Xiao-Hui, Peng, Jian-Chun, Zhan, Lin-Sheng
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Sprache:eng
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Zusammenfassung:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-β promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.
ISSN:1007-9327
2219-2840
DOI:10.3748/wjg.v16.i44.5582