Hepatitis C virus ( HCV) encodes a viral protease, nonstructural (NS) 3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(Delta 4AB) SEAP, a reporter consisting of enhanced green fluorescent protein ( EG), the NS3-NS4A protease decapeptide recognition sequence (Delta 4AB), and secreted alkaline phosphatase ( SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293E-EG( Delta 4AB) SEAP-NS3/4A, which constitutively expresses EG(Delta 4AB) SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.
