title: Inhibition of native hepatitis C virus replicase by nucleotide and non-nucleoside inhibitors.
abstract: A number of nucleotide and non-nucleoside inhibitors of HCV polymerase are currently under investigation as potential antiviral agents to treat HCV-infected patients. HCV polymerase is part of a replicase complex including the polymerase subunit NS5B together with other viral and host proteins and viral RNA. The RNA synthesis activity of the native replicase complex was inhibited by 3'-deoxy-CTP, a chain-terminating nucleotide analog, but not inhibited by non-nucleoside NS5B polymerase inhibitors of three different structural classes. The HCV replicase was also resistant to heparin, a broad-spectrum, RNA-competitive polymerase inhibitor. Prebinding of the recombinant NS5B protein with a RNA template rendered the polymerase largely resistant to the inhibition by heparin and the non-nucleoside inhibitors, but did not affect the inhibitory potency of 3'-deoxy-CTP. Therefore, the HCV replicase showed a similar pattern of inhibitor sensitivity as compared to RNA-bound NS5B. These results suggest that the native HCV replicase complex represents a stable and productive polymerase-RNA complex. The allosteric non-nucleoside NS5B polymerase inhibitors are inactive against established HCV replicase but may function antagonistically with the formation of a productive enzyme-template complex.
authors.affiliation: Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. han.ma@roche.com
authors.forename: Han
authors.initials: H
authors.lastname: Ma
authors.forename: Vincent
authors.initials: V
authors.lastname: Leveque
authors.forename: Anniek
authors.initials: A
authors.lastname: De Witte
authors.forename: Weixing
authors.initials: W
authors.lastname: Li
authors.forename: Than
authors.initials: T
authors.lastname: Hendricks
authors.forename: Saundra M
authors.initials: SM
authors.lastname: Clausen
authors.forename: Nick
authors.initials: N
authors.lastname: Cammack
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
mesh_terms: D000494:Allosteric Regulation; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase