In vivo assay for hepatitis C viral serine protease activity using a secreted protein.
Hepatitis C virus (HCV) is a major pathogen of community-acquired and post-transfusional non-A, non-B hepatitis. Since an in vitro replication system is not available, it is crucial to develop an efficient and sensitive assay system for screening inhibitors of HCV. The fact that the activity of HCV NS3 protease is responsible for the maturation of the nonstructural proteins and viral replication, suggests that NS3 protease is a suitable target for anti-HCV drug development. To devise an assay system in cell culture, we constructed NS3/4A-SEAP (secreted alkaline phosphatase) chimeric gene, in which the SEAP gene was fused in-frame to downstream of NS4A/4B cleavage site. In this system, the SEAP would be secreted into the extracellular media depending on the cleavage activity of the NS3 protease. Our results demonstrate that the NS3/4A-SEAP expression vector encoding wild type NS3 protease, but not mutant NS3 protease, could produce high SEAP activity in the media of both transfected cells and stable expression cell lines. Since the activity of SEAP in the culture media can be monitored quantitatively and continuously by the chemiluminescent method, this assay system will be useful for screening potential inhibitors of HCV protease.
Cho
Y G
YG
Department of Life Science, Center for Biofunctional Molecules, School of Environmental Engineering, Pohang University of Science and Technology, Kyungbuk, South Korea.
Yang
S H
SH
Sung
Y C
YC
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D051379:Mice; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus