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.

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 singlestranded 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.
Infections with hepatitis C virus (HCV) 3 represent a major health problem affecting ϳ2% of the world's population (1). Most patients remain chronically infected and 15-20% eventually develop liver cirrhosis and hepatocellular carcinoma. Vaccination is not available, and current therapies are associated with limited efficacy and side effects (2,3). A central issue for developing alternative antiviral strategies entails gaining a better knowledge of the molecular mechanisms governing the HCV life cycle.
Among the HCV-encoded proteins, NS5B was characterized as being the viral RNA-dependent RNA polymerase (RdRp), i.e.
a key enzyme of the viral RNA replication process and an attractive drug target (4 -6). Like other RNA polymerases, NS5B is capable of initiating RNA synthesis in the presence of a primer as well as de novo (7)(8)(9)(10)(11)(12). The available structures of NS5B display the typical right-hand architecture of polymerases consisting of "palm," "thumb," and "finger" domains (13)(14)(15). The NTP substrates are assumed to enter via a defined tunnel conformation of the protein. Modeling studies suggest that the exit of the double-stranded primer/template is blocked by a ␤-hairpin or flap (8,13), indicating that NS5B undergoes major conformational changes to accommodate the double-stranded RNA product (16).
Among a huge variety of yet characterized nucleoside and non-nucleoside inhibitors (NNI), the benzofurane derivative NNI HCV-796 was demonstrated to yield significant antiviral effects in mice with chimeric human livers and in patients infected with HCV (17). HCV-796 binds to a hydrophobic binding pocket at the "palm" domain of NS5B (18 -21); however, its mode of inhibition remains to be defined.
Although NS5B has been studied in the absence and presence of inhibitors, a firm biophysical characterization of the enzyme is lacking. To address this, 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 also enabled us to unravel the mechanism of action of HCV-796.

EXPERIMENTAL PROCEDURES
Protein Purification-The gene coding for NS5B⌬21 (HCV1b BK, including a deletion of 21 amino acids at the C terminus) was cloned into the pET SUMO vector and expressed in the Escherichia coli strain BL21(DE3) star. Biomass production was carried out using fermentation. Briefly, cells were grown at 30°C in 6 liters of medium (50 g/liter yeast extract, 0.06 M K 2 HPO 4 , 6 mM MgSO 4 , 0.03 M glucose, 0.01 M NH 4 Cl) with glycerol as carbon source (300 g/liter yeast extract, 3 M glycerol). Gene expression was induced at A 600 ϭ 50 by adding 0.8 mM isopropyl 1-thio-␤-D-galactopyranoside. Cells were harvested after 4.5 h of induction. After resuspension the cells were lysed using a French press and centrifuged at 4°C and 4.84 2 t (rotor type 45 Ti, Beckman Coulter) to remove unbroken cells and debris. Ammonium sulfate was added to the supernatant (0.5 g/ml), the resulting precipitate collected by centrifugation as above and re-dissolved. Soluble proteins were loaded on a nickel-nitrilotriacetic acid column, bound proteins were eluted by applying an imidazole gradient. After cleaving off the SUMO-protein by the SUMO-protease, a poly-(U)-Sepharose affinity chromatography step and an additional nickel-nitrilotriacetic acid chromatography step were performed, yielding untagged NS5B⌬21 with the authentic N terminus. Purified NS5B⌬21 was dialyzed against 50 mM HEPES/NaOH, 20% (v/v) glycerol, 6.5 mM MgCl 2 , 2 mM tris(2-carboxyethyl)phosphine, pH 7.5 (referred to as assay buffer) and stored at Ϫ80°C. Protein concentration was determined by measuring the absorbance at 280 nm using the extinction coefficient 83770 M Ϫ1 cm Ϫ1 .
Determination of the K D Value for the Binary Complex Composed of NS5B⌬21 and Template ssRNA by Equilibrium Fluorescence Measurements-NS5B⌬21 (HCV1b BK) was titrated to 66 nM 5ЈFAM-labeled template ssRNA (16-mer) in assay buffer at 22.5°C. Fluorescence was monitored on a Fluoromax-4 spectrofluorometer (Jobin Yvon, France). After attaining equilibrium the signals of the FAM-probed RNAs were measured (excitation at 491 nm, emission at 515 nm, and slit widths 0.2 and 5 nm) and corrected for the volume change and dynamic quenching. Relative fluorescence intensities were plotted against the protein concentration. Fitting the curves according to Equation 1 with the program KaleidaGraph TM (Synergy software) yielded the K D value of the interaction of NS5B⌬21 (HCV1b BK) and the fluorescently labeled template ssRNA. The affinity of this binary complex as a function of the ionic strength was measured in the assay buffer (with an ionic strength of 69.5 mM) at increasing NaCl concentrations and yielded apparent K D values (KЈ D ). Data were fitted according to Equation 2 and yielded the K i value of NaCl, where ⌬F is the change of normalized fluorescence, m is the concentration of the 5ЈFAM-labeled template ssRNA, n is the concentration of NS5B⌬21, and K D is native equilibrium constant between template ssRNA and HCV-NS5B. In Equation 2, KЈ D is apparent K D at the concentration of NaCl specified, K D is the native equilibrium constant between template ssRNA and HCV-NS5B in the absence of NaCl, and K i is the inhibitory constant of NaCl to template ssRNA binding to HCV-NS5B. Determination of Equilibrium Constants of the Binary Complex Composed of NS5B⌬21 and Nucleotides or HCV-796 Using CD Spectroscopy-CD spectroscopy was performed using a Jasco J-810 spectropolarimeter with the following instrumental setup: 0.5 nm data pitch, 1-s response, 20 nm/min scanning speed, 20 accumulations, 1 nm slit widths, and standard sensitivity. All experiments were carried out in assay buffer at 22.5°C. Nucleotides or the HCV-796 were titrated to 17.5 M NS5B. Spectra were recorded after attaining equilibrium and smoothed adaptively with both convolution width and deviation noise set to 5 (Spectra Manager I, Jasco). No significant spectral contributions of the buffer and nucleotides were observed. The change in the amplitude of the CD signal of the polymerase at 240 nm was directly plotted against the concentration of nucleotides or inhibitor and fitted to a sigmoidal binding behavior yielding S 0.5 and K S values of nucleotides and HCV-796, as well as the cooperativity in the absence of template ssRNA.
Fast Kinetics of Protein-RNA Complex Formation and Dissociation-The kinetics of both the association of NS5B⌬21 (HCV1b BK) and the 5ЈFAM-labeled template ssRNA (16-mer) and the dissociation of a preformed polymerase-template ssRNA complex on dilution were measured using a stoppedflow machine (SX.20 MV, Applied Photophysics) equipped with a fluorescence detection unit. The excitation of the fluorescent probe was set to 491 nm. Fluorescence emission was monitored using a cut-off filter of Ͼ515 nm. Slit widths were set to 2.2 nm each. All experiments were carried out in assay buffer at 22.5°C. Traces were fitted to a quadruple-exponential first order reaction (Equation 3) with the program Kaleida-Graph TM , yielding the respective observed rate constants. For association measurements, 78 nM NS5B⌬21 (HCV1b BK) was rapidly mixed with an equal volume of increasing amounts of the 5ЈFAM-labeled template ssRNA. Dissociation rate constants were measured by a 1:2 dilution (mixing ratio 1 ϩ 1) of 78 nM protein and 210 nM 5ЈFAM-labeled template ssRNA, preincubated in assay buffer to allow complex formation, where ⌬F is the total change of the relative fluorescence amplitude, v, x, y, and z are signal amplitudes of the respective phases, kЈ v , kЈ x , kЈ y , and kЈ z are the observed first order rate constants of the respective phases, t is time (seconds), and n is the relative fluorescence intensity at the end point of the reaction (offset).
Fast Kinetics of RNA-dependent RNA Polymerization and Product Release Reaction-To measure the incorporation of nucleotides and subsequent release of the RNA products by the HCV-RdRp, 78 nM NS5B⌬21 (HCV1b BK) and 210 nM 5ЈFAMlabeled template ssRNA (16-mer) were preincubated in assay buffer to allow the binary complex to form. Polymerase reactions were started by the addition and rapid mixing of an equal volume of increasing concentrations of NTP(s) in the stoppedflow machine. When investigating the effect of the HCV-796 on the RdRp reaction, the inhibitor was first preincubated to the preformed protein-template ssRNA complex. Afterward the reaction was started by adding 3 mM NTP(s). Because the inhibitor was dissolved in DMSO, all experiments were carried out in 1.1% (v/v) DMSO. Kinetics were monitored as fluorescence change on excitation of the FAM probe at 491 nm using a cutoff filter of Ͼ515 nm for emission. Slit widths were set to 2.2 nm each. Traces were fitted to a quadruple-exponential first order reaction according to Equation 3 considering the positive amplitude sign. Four rate constants were obtained. For data investigation using the program KaleidaGraph TM the rate constants were plotted either against the concentration of nucleotides or the inhibitor. All experiments were carried out at 22.5°C.
HCV-NS5B Polymerase Assay-The assay to determine HCV-RdRp activity was performed in a total volume of 40 l containing 0.04 M NS5B⌬21 (HCV1b BK), 3 mM each of ATP, GTP, and UTP, and 0.0825 M of [␣-32 P]CTP (3000 Ci mmol Ϫ1 , Hartmann Analytic GmbH, Braunschweig, Germany) and 0.2 M of 5ЈFAM-labeled template ssRNA in the assay buffer established. HCV-796 at concentrations of 5 M and 10 M was added to the sample in the inhibition test. The samples were incubated for 2 h at 22°C. Recovery of the radioactively labeled RNA was performed by using phenol/chloroform extraction followed by ethanol precipitation. RNA was re-suspended in formamide buffer containing 50% formamide, 5 mM EDTA, and dye marker (0.05%), pH 8.0, and separated on a denaturing polyacrylamide gel (6.5 M urea, 12% polyacrylamide in 89 mM Tris base, 89 mM boric acid, 2 mM EDTA). Autoradiography analysis was performed by phosphorimaging.

RESULTS
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). For quantitative mechanistic studies it was crucial to generate the protein in a well defined association state and devoid of any contaminating nucleic acids or nucleotides. Accordingly, a new optimized expression and purification scheme was established purifying NS5B⌬21 to homogeneity (supplemental Fig. S1; see "Experimental Procedures").
The absorbance spectrum of the purified recombinant NS5B⌬21 displayed a ratio of absorbance 280 nm/260 nm of ϳ3, clearly indicating the absence of any contaminating nucleic acids or nucleotides (supplemental Fig. S2). Analytical ultracentrifugation of NS5B⌬21 demonstrated its initial monomer state in the assay buffer used for all measurements (supplemental Fig. S3).
Binding the Template ssRNA to NS5B⌬21-Development of the RNA polymerase activity of NS5B requires binding of the two substrates, namely the template ssRNA and nucleotides. To characterize enzyme-template ssRNA binding and polymerase activity, we established a novel assay system applying 5ЈFAM fluorescently labeled ssRNA oligonucleotides as templates. Importantly, all tested RNAs enabled effective binding and de novo initiation of RNA synthesis. That is, 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. The affinity of NS5B⌬21 to fluorescently labeled substrate polymers was determined by monitoring the FAM fluorescence quenching upon binding to the protein (Fig.  1A). Thus, data analysis assuming a single binding site yielded K D values in the nanomolar range; for example, with the RNA 16-mer, it was determined to be 0.01 M ( Table 1). The fact that a low K D value was measured irrespective of whether the template contained virus-specific sequence elements demonstrated that binding of NS5B⌬21 to the RNA occurred at high affinity without template specificity. Apparent discrepancies between these results with those in earlier reports that measured different K D values of NS5B⌬21 and short template ssRNA might be explained by differences in the mode of protein preparation as well as interfering ionic effects. To investigate further these discrepancies, the K D value was measured at increasing concentrations of NaCl in the assay buffer. Starting at an ionic strength of 69.5 mM of the assay buffer itself and increasing the ionic strength by adding NaCl resulted in a linear increase of the apparent K D value (KЈ D in Equation 2). Thus, NaCl competitively inhibits the template ssRNA binding to the polymerase with a K i of 3.0 mM under the experimental conditions used (Fig. 1B).
Binding the Nucleotides to NS5B⌬21-Nucleotides are the other substrate of NS5B necessary to perform the polymerization reaction. Analyzing the NTP(s) binding by CD, near-UV CD spectra of NS5B⌬21 displayed a characteristic positive band in the absorbance range of the aromatic amino acids. Upon binding the NTP(s), the dichroic intensity of the protein decreased at 240 nm and at ϳ260 nm ( Fig. 2A). Because tertiary structural properties of proteins are reflected by near-UV CD, we interpreted the observed changes of NS5B⌬21 upon NTP binding as conformational changes in the protein. The change in dichroic signals on titration of NTP(s) to the enzyme in the absence of template ssRNA revealed the affinity of the respective nucleotides as being in the micromolar range ( Fig. 3 and Table 1). It should be noted that the data displayed a sigmoidal binding behavior reflecting a positive cooperativity throughout binding of the NTP(s) to the enzyme (see "Discussion").
The Kinetics of NS5B Comprises Two Half-reactions-The overall enzymatic reaction of NS5B proceeds as a two-substrate reaction that involves binding of the template ssRNA and the NTP(s) followed by an interaction of the two bound substrates. Substrate turnover then results in the formation of dsRNA and inorganic diphosphate as products.
In contrast to classic discontinuous assays that measure incorporation of radioactively labeled NTP(s) into dsRNA (4), here we used a continuous fluorometric assay, which enabled detailed mechanistic studies of the polymerase. Binding of template ssRNA to NS5B⌬21 led to a fluorescence decrease (quenching), whereas release of either ssRNA and/or dsRNA caused a fluorescence increase (de-quenching) (Fig. 4).
As equilibrium measurements could not be performed with the ternary complex consisting of NS5B⌬21 and the two substrates, kinetic analysis was used to determine the K m value of NTP(s). Incubation of the preformed enzyme-template ssRNA complex with nucleotides resulted in an increase of the fluores-   cence signal due to formation and release of dsRNA (Fig. 4). A kinetic analysis of this reaction in the presence of different concentrations of NTP(s) yielded a K m value of 15 M. This value is smaller by a factor of 3-6 compared with the affinity of the different nucleotides (Table 1), indicating a functional communication between the two substrate-binding sites within the enzyme. Next, we applied the time course of fluorescence intensity changes during overall substrate turnover to determine the rate constants of individual reaction steps (supplemental Fig. S4, A  and B). In accordance with reports indicating that contacts between the HCV-RdRp and the template ssRNA change during different steps in RNA-dependent RNA synthesis (10, 23), binding of the labeled template ssRNA to NS5B⌬21 in the absence of NTP(s) displayed a complex kinetic behavior. Fitting of the progress curves derived four rate constants for the stepwise binding process ( Fig. 4 and Table 2). None of the observed reactions was dependent on the concentration of template ssRNA indicating that the initial bimolecular binding reaction was too fast to be monitored (supplemental Fig. S4A). Only the subsequent reactions that led to an equilibrium in complex formation were detectable. In turn, dissociation of this complex, initiated by dilution, was also shown to proceed as a stepwise process involving four phases (Fig. 4, supplemental Fig. S4B, and Table 2).
In the second half-reaction, product formation and release were analyzed by adding NTP(s) to a preformed enzyme-substrate complex consisting of NS5B⌬21 and template ssRNA. The resulting fluorescence de-quenching kinetics were also fitted according to a quadruple-exponential first order reaction revealing the rate constants of the corresponding phases ( Fig.  5A and Table 2). De novo synthesis of dsRNA by NS5B⌬21 using the 5ЈFAM-labeled template ssRNA was also demonstrated by a discontinuous assay (Fig. 6).
Interestingly, at increasing NTP concentrations, all apparent rate constants of the first, second, and third phase decreased, whereas that of the fourth phase increased in a hyperbolic manner (Fig. 5A). We interpreted the three faster phases as steps directly involved in RNA-dependent RNA polymerization. The fourth phase was assumed to represent product release (see "Discussion").   Table 2.

TABLE 2 Kinetic parameters of NS5B⌬21-polymer binding and release
The rate constants listed and indicated with a prime represent observed rate constants (according to Scheme 1) determined by fitting progress curves of quenching and de-quenching the FAM fluorescence in NS5B⌬21 reactions according to a quadruple-exponential first order reaction, respectively. The reactions were initiated by a rapid 1 ϩ 1 mixing of the reactants and stopped-flow measurements performed as described under "Experimental Procedures." Errors of the kinetic measurements were in the range of 10%.  Inhibitor HCV-796: Mechanism of Action on Substrate Turnover-Taking advantage of the tools established here, we wanted to examine next the action of the potent NNI inhibitor HCV-796 to understand whether this compound affects the binding kinetics of the RNA-template and/or the release kinetics of the product during NTP turnover. First, by titrating the inhibitor to NS5B⌬21, we determined an IC 50 value of 0.03 mM (Fig. 3 and Table 1). No spectral contribution of HCV-796 itself in the CD analyses was detected. Performing binding assays with template ssRNA and NS5B⌬21 at saturating concentrations of HCV-796 we found that the compound did not interfere with template binding in the absence of NTP(s) ( Table 1).
For kinetic measurements of product formation and release, we applied a preformed complex composed of NS5B⌬21, template ssRNA, and varying concentrations of HCV-796. By adding NTP(s) at saturating concentrations, again four phases of release were monitored, and, at increasing inhibitor concentrations, all phases were accelerated in a hyperbolic manner ( Fig.   5B and Table 2). It is important to note that, at saturating concentrations of HCV-796 and NTP(s), the discontinuous radioactive assay revealed no dsRNA formation (Fig. 6). Thus, acceleration of the observed phases was concluded to reflect an augmented template ssRNA release. Fitting the rate constants in dependence of the inhibitor concentration yielded a K i value of ϳ7 M for the inhibitor. This result was congruent with data obtained when testing the inhibitor in the radioactive discontinuous assay performed at concentrations of 5 M and 10 M, respectively (Fig. 6). Hence, it can be stated that HCV-796 interferes with dsRNA formation by blocking the second halfreaction. Importantly, no binding of the template ssRNA to NS5B⌬21 was measured when preincubated with HCV-796 and NTP(s) at saturating concentrations.

DISCUSSION
Mechanistic Aspects of NS5B Action-The main aim of this work was to characterize the HCV-RdRp in terms of substrate binding, turnover, and product release. As with other polymerases (24 -27), the entire enzymatic reaction can be characterized as a two-substrate reaction, namely the incorporation of FIGURE 5. Determination of the K m value for NTP(s) and the K i value for HCV-796 affecting HCV-RdRp NS5B⌬21. A, the progress curves of 5ЈFAMlabeled dsRNA formation were fitted according to quadruple-exponential first order reactions. The first (circle), second (square), and third (diamond) phases are decelerated with increasing nucleotide concentration in a hyperbolic manner, whereas the fourth (cross) phase is accelerated. B, the progress curves of 5ЈFAM-labeled RNA release in dependence of the inhibitor concentration and at saturating concentrations of the substrates were fitted according to quadruple-exponential first order reactions. Note that all four phases (labels as mentioned above) are accelerated with increasing HCV-796 concentration in a hyperbolic manner. The enzymatic parameters are summarized in Tables 1 and 2. The reaction conditions were performed as described under "Experimental Procedures." FIGURE 6. Enzymatic formation of dsRNA by HCV-RdRp NS5B⌬21 and its inhibition by HCV-796 using 5FAM-labeled template ssRNA (16-mer) as substrate in the discontinuous radioactive polymerase assay. The test was performed according to previous studies (4,5) with slight modifications. The reaction conditions were performed as described under "Experimental Procedures." nt, nucleotides.
nucleotides to a substrate-polymer on the basis of complementary base pairing. Using a fluorescently labeled template ssRNA and monitoring the course (kinetics) of the enzymatic reaction in a continuous assay and by CD spectroscopy we demonstrated that the template ssRNA and the NTP(s) are capable of binding independently to the enzyme. Importantly, the binding constants for both substrates turned out to be rather different. The K D values of the applied template ssRNAs were in the nanomolar range, whereas the K D values of the respective NTP(s) turned out to be in the micromolar range (Table 1). Interestingly, the K m value of the NTPs indicated that nucleotide binding improved as a result of template ssRNA binding during the course of the entire enzymatic reaction (Table 1). These data are reasonable in view of the physiological situation of the cell where nucleotides are present at saturating concentrations. We therefore concluded that substrate turnover proceeds according to a two-substrate reaction composed of two half-reactions. The binding reaction of both substrates covers the first half-reaction of the overall sequential reaction mechanism (formation of the ternary complex).
Equilibrium binding of NTP(s) results in conformational changes of the enzyme. This became apparent in far-and near-UV CD by measuring chirality changes (Fig. 3). By fitting the observed sigmoidal binding behavior, we obtained Hill coefficients indicating a positive cooperativity of nucleotide binding. One explanation for this interesting observation is the existence of an additional binding site for nucleotides to NS5B. Such a scenario has been discussed previously in the context of an allosteric regulation or additional docking site of NTP(s) (28 -30). Due to their analogous chemical nature there are generally two different binding sites present in this protein, namely for the template ssRNA and the nucleotides. In the absence of template ssRNA, the NTP(s) will occupy both sites in a cooperative manner. Accordingly, binding of GTP as well as of the other nucleotides to HCV-NS5B revealed a Hill coefficient of ϳ2. Thus, association of a nucleotide to the NTP site is accompanied by nucleotide binding to the substratepolymer site.
Interestingly, as measured by phased fluorescence quenching curves (Fig. 4), attachment and incorporation of the template ssRNA to the active site of NS5B were found to proceed in a stepwise manner. We interpreted this observation as a positioning process of the template ssRNA within the active site that is trapped via several intermediates. The same behavior was observed in the release reaction of the template where we measured a phased dye fluorescence dequenching (Scheme 1).
Binding of the two substrates by NS5B⌬21 in the first half-reaction occurs in a random manner (random bi-bi mechanism). Thus, considering again the difference in the K D values of the template ssRNA and NTP(s) by some orders of magnitude, a fairly ordered mechanism is conceivable. With the ternary complex as a starting point, the second halfreaction then covers the formation and release of dsRNA. Along this line, the complex kinetics involving at least four kinetically detectable phases (Table 2) reflect a stepwise formation/positioning of the product within the active site and a final release, respectively. Moreover, product formation by NS5B⌬21 acting on the fluorescent template ssRNA was verified by a classic polymerase assay measuring the incorporation of radioactively labeled nucleotides (Fig. 6).
As already outlined, at increasing nucleotide concentrations all apparent rate constants of the first, second, and third phase decreased, whereas the rate constant of the fourth phase increased in a hyperbolic manner (Fig. 5A). Thus, at saturating concentrations, the reaction sequence of product formation/ positioning catalyzed by the enzyme is decelerated in comparison to the substrate-polymer dissociation reaction (Table 2). Apparently, at non-saturating nucleotide concentrations a partitioning occurs between product-polymer and substrate-polymer release. In sum, we conclude that the three faster phases in the RdRp reaction sequence represent reaction steps that are directly involved in RNA-dependent RNA polymerization. In other words we suggest the decrease in re-positioning rates along with increasing concentrations of NTP(s) corresponding to the actual incorporation of bound nucleotides into the dsRNA. Conversely, the fourth phase, which is accelerated in effective enzyme catalysis, is assumed to represent the product release and may thus operate as a "gatekeeper" function.
HCV-796: Mode of Inhibition-Supported by the available structural data of the HCV-RdRp, much effort has been put into the rational design of a wide variety of small molecule inhibitors that were classified according to their specific enzyme binding SCHEME 1 sites (see the introduction) (31). Applying NS5B of HCV strain Con1 and measuring the change of the intrinsic fluorescence of the enzyme upon compound binding, the potent NNI HCV-796 was recently indicated to develop "slow binding kinetics" (18). Here, by monitoring the amplitude change in CD of NS5B⌬21 when exposed to the inhibitor, a slow binding mode of action of HCV-796 could not be confirmed (Fig. 2B). Discrepancies, which may relate to different applied HCV strains, were also obtained in terms of the determined K i values. Using NS5B⌬21 of HCV1b strain BK, we established a K i value of 7 M for HCV-796, whereas the previously reported K i value of 0.07 M obtained with NS5B⌬21 of HCV1b strain Con1 was two orders of magnitude lower. It was recently reported that the potential of HCV-796 was significantly reduced with HCV species in which residue Cys-316 in close proximity to the active site of NS5B was substituted for Asn (18,19). Indeed, in comparison to the Con1 NS5B that contains Cys-316 the BK NS5B used in this study contains Asn-316.
It is important to note that HCV-796 incorporation into NS5B⌬21 resulted in a conformational switch detectable by chirality changes. Moreover, CD of the binary complex revealed a distinct bathochromic shift of the negative maximum, which points to a binding mode of the inhibitor different from that of nucleotides (Fig. 2, A and B). Finally, these data indicate a cooperative binding of HCV-796 ( Fig. 3 and Table 1), which might come about by the stacking and binding properties of the compound and cannot be interpreted without additional structural information.
For a detailed analysis of the mode of inhibition, we determined binding and turnover of the template ssRNA in the presence of HCV-796. In the first half-reaction (formation of the ternary complex) the inhibitor did not affect the affinity of the substrate-polymer to NS5B⌬21 in the absence of nucleotides. However, the addition of NTP(s) to this ternary complex resulted in a release of the template ssRNA without product formation ( Fig. 6 and Table 1). NTP(s) indeed induce a conformational switch in NS5B⌬21, which is detectable by CD spectroscopy ( Fig. 2A). In the presence of the allosteric inhibitor HCV-796 this nucleotide-induced conformational change dramatically impairs the enzyme's affinity for the template ssRNA. Additionally, this became evident when analyzing the rate constants of the RdRp reaction at increasing concentrations of HCV-796 ( Fig. 5B and Table 2).
In the productive RdRp reaction (second half-reaction) the three faster phases were decelerated and might be related to the nucleotide incorporation, whereas the rate-limiting step was accelerated and thus represents successful dsRNA formation (Fig. 5A). Interestingly, the inhibitor HCV-796 accelerated all observed phases of template ssRNA release, but did not result in product formation (Figs. 5B and 6). Thus, the inhibitory effect of HCV-796 may have two origins. First, it prevents the incorporation of nucleotides. At increasing inhibitor concentrations the observed rate constants of the three faster phases increased due to the release of template ssRNA. Second, the rate-limiting step was also accelerated. Hence, HCV-796 cancels out the gatekeeper function by impairing the ability of the enzyme to discriminate between ssRNA and dsRNA.
Taken together, the procedures established in this study to measure the kinetics of the HCV-RdRp proved to be valuable tools for a further analysis of the activity of modulating viral and cellular proteins as well as for the further evaluation of anti-HCV compounds.