A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells

Abstract

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EG(Δ4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(Δ4AB)SEAP. EG(Δ4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Δ4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of Δ4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(Δ4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(Δ4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(Δ4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(Δ4AB)SEAP cells. In conclusion, Ava5-EG(Δ4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.