Home HOME Acta Virologica 2008 Acta Virologica Vol.52, No.3, p.133-141, 2008

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Quarterly,
Founded: 1957
ISSN 0001-723X
E-ISSN 1336-2305

Published in English

Impact Factor = 1.82

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Acta Virologica Vol.52, No.3, p.133-141, 2008

Title: DEVELOPMENT OF A CELL-BASED ASSAY FOR MONITORING HEPATITIS C VIRUS NS3/4A PROTEASE ACTIVITY
Author: Y.F. LEI, W. YIN, J. YANG, X. LV, S.H. WEI, Q.X. AN, X.B. HU, Z.K. XU
Abstract: Hepatitis C virus (HCV) NS3/4A (non-structural 3 and 4 B) protease plays a key role in the processing of polyprotein precursor and it becomes an attractive target for antiviral drug discovery. We developed a cell-based assay for monitoring of the NS3/4A protease activity in mammalian cells that is an important step in screening of specific drugs against the protease. The recombinant caspase 3 (rCasp3) was used as the specific substrate for NS3/4A protease. The endogenous cleavage sites in the procaspase 3 molecule were substituted by decapeptides specific for NS3/4A protease. The activation of rCasp3 depended on its specific cleavage by NS3/4A protease and resulted in an apoptosis of stable cells expressing the protease. The difference in cell viability between the cells expressing NS3/4A protease transfected with rCasp3 and the counterparts pretreated with NS3/4A protease inhibitors could be estimated by a spectrophotometry based on 3-(4,5-dimethylthioazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining of cells in microplates. Thus, we developed a simple and cost-effective colorimetric assay for evaluating NS3/4A protease activity enabling the screening of candidate NS3/4A protease inhibitors.
Keywords: apoptosis; hepatitis C virus; recombinant Casp3; MTT assay; NS3/4A protease
Year: 2008, Volume: 52, Issue: 3 Page From: 133, Page To: 141
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