High-Throughput Screening and Rapid Inhibitor Triage Using an Infectious Chimeric Hepatitis C Virus

Abstract

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

“…Recently, several groups have reported cell-based HCV infection systems for the screening of HCV inhibitors in various assay formats (12)(13)(14)(15). Traditionally, HTS was performed at a single fixed concentration of each candidate compound, which is prone to false positives and false negatives and requires extensive follow-up studies.…”

“…Further studies demonstrated that reporter genes can be genetically engineered into certain locations of the HCV genome without impairing the viability of the virus (11). Several groups have reported cell-based HCV infection systems for the screening of HCV inhibitors in various assay formats (12)(13)(14)(15). Gastaminza et al developed a colorimetric assay measuring HCV E2 protein produced by HCVinfected cells in a 96-well plate format (12).…”

“…Based on the ability of the HCV NS3 protease to cleave synthetic peptides containing the enzyme's natural viral cleavage sites, Yu et al applied a cell-based hepatitis C virus infection fluorescence resonance energy transfer (FRET) assay for antiviralcompound screening (13). Using a luciferase reporter that is directly inserted into the HCV genome, Wichroski et al adapted the infection system in a 384-well format for the screening of HCV inhibitors (15).…”

Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses

“…A significant deterrent to automated anti-RABV drug discovery, for instance, is the BSL2/3 containment requirements imposed by replication-competent RABVs and mandatory rabies vaccination as well restriction to the laboratory of not vaccinated people.. Based on the original reverse genetics system developed for the SAD-B19 RABV strain [64], however, minireplicon systems and single-cycle reporter RABV viruses were developed that allow study of the RABV polymerase activity and in-cell replication in a BSL2 setting [65][66][67][68][69][70][71][72][73]. Analogous to the precedent set by successful screens employing single-cycle HIV, hepatitis C virus, and influenza virus reporter strains [74][75][76], the single-cycle based approach in particular offers an exciting drug discovery perspective. Transient-transfection based minigenome drug screens have furthermore been attempted to identify, for instance, Ebola virus polymerase inhibitors [77][78][79].…”

Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay

“…Reporter genes inserted into certain locations of the HCV genome could yield viable HCV capable of infection and replication in the cell culture (Koutsoudakis et al, 2006). In recent years, several groups have applied either wild-type HCV or modified HCV with reporter genes to develop cell-based HCV infection systems for the screening of HCV inhibitors (Chockalingam et al, 2010; Gastaminza et al, 2010; Kim et al, 2007; Wichroski et al, 2012; Yu et al, 2012). Cell-based HCV infection systems have the advantage of targeting various stages of HCV life cycle, thus significantly improving the efficiency of the drug screening process.…”