High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

Abstract

Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

“…2A) into cells resulted in dual-phase luciferase expression. While the first phase of luciferase expression peaked at 3 h posttransfection and declined to the background level at 10 h posttransfection, the second phase of expression started at 24 h posttransfection and increased exponentially (39). It had been demonstrated that the first phase of luciferase expression was translated from the input replicon RNA, but the second phase of luciferase expression required replicon RNA replication (39).…”

“…To identify ISGs that are able to inhibit WNV and/or DENV infections, we initially examined the effects of each individual ISG on WNV and DENV-1 replicon-containing virus-like particle (VLP) infections in FLP-IN T Rex-derived ISG-expressing cell lines. As described previously (39), WNV and DENV-1 VLPs were prepared by providing the viral structural proteins in trans in cells containing viral replicons. Like virions, the VLPs are able to infect permissive cells and initiate replicon RNA replication.…”

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

“…Finally, toxic compounds can be scored as positive hits, and therefore must be eliminated with a counter screen measuring cellular viability. To overcome some of these hurdles, recombinant viruses or replicons have been engineered by reverse genetics to express reporter proteins, such as EGFP or luciferase, from an additional transcription unit or in frame with viral protein genes (few examples are [20][21][22][23] ). When these viruses replicate, reporter proteins are produced together with viral proteins themselves.…”

A Computational Technique to Estimate Within-Host Productively Infected Cell Lifetimes in Emerging Viral Infections

“…The WNV replicon was genetically engineered to encode a Renilla luciferase reporter and had the genes encoding for WNV structural proteins deleted. Viral structural proteins were packaged in trans to create virus-like particles as described in [10]. The WNV replicon was injected subcutaneously into the footpad of six week old B6 mice.…”