AI Article Synopsis
- The hepatitis C virus (HCV) forms replication complexes on the endoplasmic reticulum that can be visualized as "dot-like" structures using fluorescence microscopy.
- To identify compounds that inhibit the formation of these complexes, researchers developed a high-content screening assay in a specialized cell line that allows for controlled expression of HCV proteins.
- The assay proved effective, as demonstrated by a dose-dependent decrease in "dot-like" structures when a specific HCV protease inhibitor was applied.
Article Abstract
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.
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| http://dx.doi.org/10.1016/j.jviromet.2010.02.007 | DOI Listing |
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