Development of a high-content screening assay to identify compounds interfering with the formation of the hepatitis C virus replication complex.

The hepatitis C virus (HCV) replicates its genome on a membrane-associated replication complex. These complexes are represented by "dot-like" structures on the endoplasmic reticulum when standard fluorescence microscopy techniques are applied. To screen compound libraries for inhibitors interfering with the formation of the HCV replication complex independent of RNA replication, an image-based high-content screening assay was developed utilizing inducible expression of the HCV non-structural proteins NS3-5B in an U2-OS Tet-On cell line. An eGFP was fused to NS5A for the detection of replication complexes. The cell line was tightly regulated and the eGFP insertion within NS5A did not alter polyprotein processing. The NS5AeGFP signal colocalized with other non-structural proteins in "dot-like" structures. Accompanying image analysis tools were developed enabling the detection of changes in replication complex formation. Finally, the addition of a HCV NS3/4A protease inhibitor resulted in a dose-dependent reduction of "dot-like" structures demonstrating the practicability of the assay.