title: Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.
abstract: Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.
authors.affiliation: Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research; a joint venture between Medical School Hannover (MHH) and Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Straße 7-9, 30625 Hannover, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Gentzsch
authors.forename: Bettina
authors.initials: B
authors.lastname: Hinkelmann
authors.forename: Lars
authors.initials: L
authors.lastname: Kaderali
authors.forename: Herbert
authors.initials: H
authors.lastname: Irschik
authors.forename: Rolf
authors.initials: R
authors.lastname: Jansen
authors.forename: Florenz
authors.initials: F
authors.lastname: Sasse
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.forename: Thomas
authors.initials: T
authors.lastname: Pietschmann
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006133:Growth Substances / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D006133:Growth Substances