摘要
AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS: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.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom 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.CONCLUSION: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.
AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS: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.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom 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.CONCLUSION: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.
基金
Supported by The Natural Science Foundation of China,No.30600330,No.30671842,No.30672488,No.30700475,No.30771919and No.30700757
the National High Technology Research and Development Program of China,No.2008AA02Z132
Beijing Municipal Natural Science Foundation,No.5082016
Mega-projects of Science Research for the 11th Five-Year Plan,No.2009ZX10004-4001
