title: Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides.
abstract: The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.
authors.affiliation: Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia, Italy.
authors.forename: Licia
authors.initials: L
authors.lastname: Tomei
authors.forename: Sergio
authors.initials: S
authors.lastname: Altamura
authors.forename: Linda
authors.initials: L
authors.lastname: Bartholomew
authors.forename: Monica
authors.initials: M
authors.lastname: Bisbocci
authors.forename: Carolyn
authors.initials: C
authors.lastname: Bailey
authors.forename: Michele
authors.initials: M
authors.lastname: Bosserman
authors.forename: Antonella
authors.initials: A
authors.lastname: Cellucci
authors.forename: Eleonora
authors.initials: E
authors.lastname: Forte
authors.forename: Ilario
authors.initials: I
authors.lastname: Incitti
authors.forename: Laura
authors.initials: L
authors.lastname: Orsatti
authors.forename: Uwe
authors.initials: U
authors.lastname: Koch
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: David B
authors.initials: DB
authors.lastname: Olsen
authors.forename: Steven S
authors.initials: SS
authors.lastname: Carroll
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology*; D001581:Benzothiadiazines / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D001562:Benzimidazoles; D001581:Benzothiadiazines; D004791:Enzyme Inhibitors; D012367:RNA, Viral; C031000:benzimidazole; D012324:RNA-Dependent RNA Polymerase