title: Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells.
abstract: The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.
authors.affiliation: Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei 115, Taiwan, Republic of China.
authors.forename: Jin-Ching
authors.initials: JC
authors.lastname: Lee
authors.forename: Ya-Feng
authors.initials: YF
authors.lastname: Shih
authors.forename: Sung-Po
authors.initials: SP
authors.lastname: Hsu
authors.forename: Ten-Yuan
authors.initials: TY
authors.lastname: Chang
authors.forename: Lee-Hua
authors.initials: LH
authors.lastname: Chen
authors.forename: John T A
authors.initials: JT
authors.lastname: Hsu
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000096:biosynthesis; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008826:Microbial Sensitivity Tests; D012093:Replicon; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D014764:Viral Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D002352:Carrier Proteins; D003470:Culture Media; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D016898:Interferon-alpha; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D001616:beta-Galactosidase; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus