Mechanisms of activity and inhibition of the hepatitis C virus RNA-dependent RNA polymerase.
The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.
Reich
Stefan
S
Department of Microbial Biotechnology, Institute of Biochemistry and Biotechnology, Faculty of Life Sciences, Martin-Luther-University Halle-Wittenberg, D-06120 Halle/Saale, Germany.
Golbik
Ralph Peter
RP
Geissler
René
R
Lilie
Hauke
H
Behrens
Sven-Erik
SE
D001572:Benzofurans / Q000737:chemistry* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry* / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology
D001572:Benzofurans; D004791:Enzyme Inhibitors; C539481:HCV 796; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; D013449:Sulfonamides; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase