Inhibition of native hepatitis C virus replicase by nucleotide and non-nucleoside inhibitors.
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.
Ma
Han
H
Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA. han.ma@roche.com
Leveque
Vincent
V
De Witte
Anniek
A
Li
Weixing
W
Hendricks
Than
T
Clausen
Saundra M
SM
Cammack
Nick
N
Klumpp
Klaus
K
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
D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase