Abstract
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.
Highlights
Among the hepatitis C virus (HCV)-encoded proteins, NS5B was characterized as being the viral RNA-dependent RNA polymerase (RdRp), i.e
Preparation of HCV-NS5B⌬21—Based on earlier reports, we used in the current study the NS5B⌬21 variant lacking the C-terminal amphipathic helix that is involved in membrane anchoring but is not essential for RdRp activity [22]
We found no significant differences when testing RNAs corresponding to the 3Ј-end of the HCV negative-strand intermediate, which was utilized as a preferred substrate of the polymerase [18], and a randomly composed 16-nucleotide RNA that was primarily used in this study
Summary
Among the HCV-encoded proteins, NS5B was characterized as being the viral RNA-dependent RNA polymerase (RdRp), i.e. Grant RO1DK062847 3 The abbreviations used are: HCV, hepatitis C virus; dsRNA, double-stranded RNA; HCV-RdRp, RNA-dependent RNA polymerase of HCV; NNI, non-nucleoside inhibitor; NS5B, non-structural protein 5B; NS5B⌬21, NS5B with C-terminal deletion of 21 amino acids; NTP(s), nucleotide(s) (mixture containing ATP, GTP, CTP, and UTP at equimolar concentration); ssRNA, singlestranded RNA. We have purified the protein with a high quality and established a new assay system, which has allowed a quantitative characterization of binding and substrate turnover by rapid transient kinetic methods. This system enabled us to unravel the mechanism of action of HCV-796
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