High-Throughput Screening of HCV RNA Replication Inhibitors by Means of a Reporter Replicon System

Abstract

Efforts to find effective treatment for hepatitis C virus (HCV) have been hampered by the lack of a robust in vitro infectious tissue-culture system for this virus. A subgenomic replicon system was first developed in 1999 and has since been extensively optimized to accommodate the need for conveniently measuring HCV replication in vitro and widely adopted in HCV drug-discovery efforts. Here we describe the adaptation of a modified replicon system for a high-throughput screening (HTS) in anti-HCV drug discovery. In this system, the antiviral activity and cytotoxicity of any experimental compound are measured from a single well. This duplex measurement greatly increases the efficiency of the HTS while lowering the cost. The usefulness of this approach has been supported by the recent discovery of many new lead compounds from our HTS efforts in the past two years.

Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

“…Several chimeric JFH1 reporter viruses have been developed for analyzing virus infection (1,15,22,25,39,50). One common strategy is to modify HCV cDNA by inserting GFP or a luciferase gene into the viral genome in a monocistronic or bicistronic reporter construction.…”

“…Cellular and viral proteases are involved in processing viral polyprotein into at least 10 proteins (core, E1, E2, p7, nonstructure protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B), and the cleavages at the NS3-4A, NS4A-4B, NS4B-5A, and NS5A-5B junctions are mediated by NS3/4A protease (13,24,26). In the past few years, subgenomic HCV replicons have been employed extensively for studying viral replication, protein processing, and virus-host interactions and for discovering anti-HCV agents (4,15,16,29,34,52). However, such subgenomic systems are not useful for studying the entry, assembly, or release of viral particles, because they lack structure proteins.…”

“…Therefore, a highthroughput screening system for accelerating the discovery and development of anti-HCV therapeutics is urgently needed. Recombinant JFH1 viruses that contain a luciferase or green fluorescent protein (GFP) reporter gene have recently been generated to investigate viral infection (1,15,25,50). However, the associated common problem for these constructions is the low yield of viral progeny, possibly due to the insertion of undesirable elements that impair RNA replication.…”

Development and Evaluation of a FACS-Based Medium-Throughput Assay for HCV Entry Inhibitors

“…For this reason, several high-throughput screening assays (e.g., for HCV RNA replication inhibitors) have been developed that are aimed at finding novel therapeutic approaches to HCV treatment. 28 So far, no screening system for HCV entry inhibitors has been published. Regarding HIV, there are promising screening systems published for entry inhibitors leading to hit compounds inhibiting cell-cell fusion in the lowmicromolar range.…”

High‐throughput screening (HTS) for the identification of novel antiviral scaffolds

“…Another virus of concern from the same family is dengue virus. So, it would be worthwhile to use the knowledge generated and the strategies gained from the successful drug development process for HCV for the discovery of antiviral agent for dengue. Dengue fever is the most frequent arthropod‐borne viral disease of humans, with almost half of the world's population at risk.…”