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• W2045880555 abstract "Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2′-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2′-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step (∼150-fold) compared with ∼2-fold at the elongation step. Interestingly, discrimination of the 2′-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the “flap,” encompassing amino acids 443–454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2′-modified nucleotide analogues, because it is more permissive to inhibition than initiation. Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2′-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2′-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step (∼150-fold) compared with ∼2-fold at the elongation step. Interestingly, discrimination of the 2′-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the “flap,” encompassing amino acids 443–454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2′-modified nucleotide analogues, because it is more permissive to inhibition than initiation. The Flaviviridae is an important virus family comprising three genera, namely flavivirus, pestivirus, and hepacivirus. Both flavivirus and hepacivirus genera comprise major human pathogens, such as Dengue virus, West Nile virus, Yellow Fever virus, and the Hepatitis C virus (HCV 1The abbreviations used are: HCV, hepatitis C virus; RdRp, RNA-dependent RNA polymerase; IMPDH, inosine-5′-monophosphate dehydrogenase; HIV-1, human immunodeficiency virus, type 1; WT, wild type; IC50, inhibitory concentration of 50%. ), respectively. The discovery of HCV in the late 1980s and analysis of its prevalence in the general population has revealed that HCV is the most common etiological agent of chronic liver disease (1Wasley A. Alter M.J. Semin. Liver Dis. 2000; 20: 1-16Crossref PubMed Google Scholar). More than 170 millions persons are infected by HCV in the world, making these individuals at risk of developing liver cirrhosis and hepatocellular carcinoma (2Liang T.J. Rehermann B. Seeff L.B. Hoofnagle J.H. Ann. Intern. Med. 2000; 132: 296-305Crossref PubMed Scopus (802) Google Scholar). Unlike other viral diseases for which antiviral therapies exist (e.g. human immunodeficiency virus or herpes infections), a persistent HCV infection can be controlled (3Poynard T. Marcellin P. Lee S.S. Niederau C. Minuk G.S. Ideo G. Bain V. Heathcote J. Zeuzem S. Trepo C. Albrecht J. Lancet. 1998; 352: 1426-1432Abstract Full Text Full Text PDF PubMed Scopus (2405) Google Scholar, 4McHutchison J.G. Gordon S.C. Schiff E.R. Shiffman M.L. Lee W.M. Rustgi V.K. Goodman Z.D. Ling M.H. Cort S. Albrecht J.K. N. Engl. J. Med. 1998; 339: 1485-1492Crossref PubMed Scopus (3361) Google Scholar). In some cases, HCV can be eradicated from an infected patient (5Somsouk M. Lauer G.M. Casson D. Terella A. Day C.L. Walker B.D. Chung R.T. Gastroenterology. 2003; 124: 1946-1949Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and this represents the first example of a complete success of antiviral therapy ever. Current antiviral therapies rely on the association of interferon α to the nucleoside analogue ribavirin. Ribavirin is a broad spectrum antiviral agent discovered about 30 years ago. Although not effective on all HCV isolates and associated with undesirable side effects (6Lauer G.M. Walker B.D. N. Engl. J. Med. 2001; 345: 41-52Crossref PubMed Scopus (2505) Google Scholar), ribavirin exerts its antiviral activity by at least two mechanisms that are intimately linked. First, it is intracellularly converted to ribavirin 5′-monophosphate, which is a potent inhibitor of the cellular inosine 5′-monophosphate dehydrogenase (IMPDH) (7Miller J. Kigwana L. Streeter D. Robins R. Simon L. Roboz J. Ann. N. Y. Acad. Sci. 1977; 284: 211-229Crossref PubMed Scopus (76) Google Scholar, 8Streeter D.G. Witkowski J.T. Khare G.P. Sidwell R.W. Bauer R.J. Robins R.K. Simon L.N. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1174-1178Crossref PubMed Scopus (451) Google Scholar). Inhibition of IMPDH depresses the intracellular concentration of GTP. Second, ribavirin is also converted to ribavirin triphosphate and acts as a GTP analogue. It is incorporated into viral RNA, leading to lethal mutagenesis of the viral genome (9Vo N.V. Young K.C. Lai M.M. Biochemistry. 2003; 42: 10462-10471Crossref PubMed Scopus (72) Google Scholar) and inhibits the polymerase directly after its incorporation (9Vo N.V. Young K.C. Lai M.M. Biochemistry. 2003; 42: 10462-10471Crossref PubMed Scopus (72) Google Scholar, 10Maag D. Castro C. Hong Z. Cameron C.E. J. Biol. Chem. 2001; 276: 46094-46098Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 11Gallois-Montbrun S. Chen Y. Dutartre H. Sophys M. Morera S. Guerreiro C. Schneider B. Mulard L. Janin J. Veron M. Deville-Bonne D. Canard B. Mol. Pharmacol. 2003; 63: 538-546Crossref PubMed Scopus (25) Google Scholar). Thus, IMPDH inhibition and subsequent depression of cellular GTP pools potentiate the action of ribavirin triphosphate as an antiviral nucleotide analogue. The antiviral action of ribavirin as a nucleotide analogue indicates not only that the HCV polymerase is an interesting target for antiviral drugs but also that nucleosides in general are interesting molecules to develop potent anti-HCV treatments. Therefore, several nucleoside analogues are currently being evaluated as potential anti-HCV agents, such as 2′-O-methyl nucleosides, 2′-C-methyl nucleosides, and β-d-N4-hydroxycytidine (12Schinazi R.F. Larder B.A. Mellors J.W. Int. Antiviral News. 2000; 8: 65-91Google Scholar, 13Migliaccio G. Tomassini J.E. Carroll S.S. Tomei L. Altamura S. Bhat B. Bartholomew L. Bosserman M.R. Ceccacci A. Colwell L.F. Cortese R. De Francesco R. Eldrup A.B. Getty K.L. Hou X.S. LaFemina R.L. Ludmerer S.W. MacCoss M. McMasters D.R. Stahlhut M.W. Olsen D.B. Hazuda D.J. Flores O.A. J. Biol. Chem. 2003; 278: 49164-49170Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 14Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), or canonical 3′-deoxyribonucleotides (15Shim J. Larson G. Lai V. Naim S. Wu J.Z. Antiviral Res. 2003; 58: 243-251Crossref PubMed Scopus (52) Google Scholar). A clear picture of the mechanism of action of some of these inhibitors is beginning to emerge. In the case of β-d-N4-hydroxycytidine, no direct inhibition of the polymerase is observed using the purified enzyme in assays in vitro (12Schinazi R.F. Larder B.A. Mellors J.W. Int. Antiviral News. 2000; 8: 65-91Google Scholar). Rather, it is thought that the analogue 5′-monophosphate is incorporated into the viral RNA selectively leading to biologically inactive genomes. In the case of 2′-modified analogues, it has been reported that they act as non-obligate chain terminator of RNA synthesis (14Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Unlike obligate chain terminators (e.g. azidothymidine, 2′,3′-dideoxynucleosides in antiretroviral treatments), non-obligate chain terminators carry a 3′-hydroxyl group, and thus bear potential to support further RNA synthesis once incorporated into viral RNA. Most of our understanding of the mechanism of action of nucleoside analogues comes from studies performed on the HIV-1-reverse transcriptase and its nucleoside inhibitors (for recent reviews, see Refs. 16Menendez-Arias L. Trends Pharmacol. Sci. 2002; 23: 381-388Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar and 17Deval J. Courcambeck J. Selmi B. Boretto J. Canard B. Curr. Drug Metab. 2004; 5: 305-316Crossref PubMed Scopus (27) Google Scholar). Reverse transcriptase is a DNA and RNA primer-dependent DNA polymerase. The HCV polymerase, however, is an RNA polymerase able to initiate RNA synthesis without primer in a so-called de novo RNA synthesis process, as well as to elongate an existing RNA primer (18Oh J.W. Ito T. Lai M.M. J. Virol. 1999; 73: 7694-7702Crossref PubMed Google Scholar, 19Luo G. Hamatake R.K. Mathis D.M. Racela J. Rigat K.L. Lemm J. Colonno R.J. J. Virol. 2000; 74: 851-863Crossref PubMed Scopus (253) Google Scholar, 20Zhong W. Uss A.S. Ferrari E. Lau J.Y. Hong Z. J. Virol. 2000; 74: 2017-2022Crossref PubMed Scopus (166) Google Scholar). Primer-independent RNA synthesis is unique to viral RNA polymerases so far. To perform such an initiation step, these polymerases have selected through evolution unique structural features essential to the synthesis of their own short RNA primers (21Choi K.H. Groarke J.M. Young D.C. Kuhn R.J. Smith J.L. Pevear D.C. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4425-4430Crossref PubMed Scopus (195) Google Scholar, 22Butcher S.J. Grimes J.M. Makeyev E.V. Bamford D.H. Stuart D.I. Nature. 2001; 410: 235-240Crossref PubMed Scopus (437) Google Scholar, 23Bressanelli S. Tomei L. Rey F.A. De Francesco R. J. Virol. 2002; 76: 3482-3492Crossref PubMed Scopus (337) Google Scholar). The crystal structure of the HCV polymerase as well as some elegant enzymatic assays have identified such a structure (23Bressanelli S. Tomei L. Rey F.A. De Francesco R. J. Virol. 2002; 76: 3482-3492Crossref PubMed Scopus (337) Google Scholar, 24Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 25Lesburg C.A. Cable M.B. Ferrari E. Hong Z. Mannarino A.F. Weber P.C. Nat. Struct. Biol. 1999; 6: 937-943Crossref PubMed Scopus (700) Google Scholar). A β-strand-turn-β-strand (“flap”) subdomain actually obstructs the polymerase active site when the latter is compared with related primer-dependent polymerases. Based on both structural and deletion studies, it has been proposed that the flap gives physical support to initiating nucleotides, up to the point where the neo-synthesized primer RNA triggers a conformational change of the polymerase active site (23Bressanelli S. Tomei L. Rey F.A. De Francesco R. J. Virol. 2002; 76: 3482-3492Crossref PubMed Scopus (337) Google Scholar, 24Hong Z. Cameron C.E. Walker M.P. Castro C. Yao N. Lau J.Y. Zhong W. Virology. 2001; 285: 6-11Crossref PubMed Scopus (175) Google Scholar, 26Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K. Cameron C.E. Lau J.Y. Hong Z. J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar). Subsequently, the polymerase must adopt an alternate conformation in which the primer is elongated in a processive fashion. Recently, the flap has been shown to play a role in repressing primer-directed RNA synthesis in favor of initiation of RNA synthesis (27Ranjith-Kumar C.T. Gutshall L. Sarisky R.T. Kao C.C. J. Mol. Biol. 2003; 330: 675-685Crossref PubMed Scopus (52) Google Scholar). The HCV polymerase has been reported to be activated by GTP, which is able to bind to a low affinity allosteric site away from the polymerase active site (23Bressanelli S. Tomei L. Rey F.A. De Francesco R. J. Virol. 2002; 76: 3482-3492Crossref PubMed Scopus (337) Google Scholar). It is suspected that the action of GTP at this allosteric site plays a role in the triggering of the switch between initiation and elongation. These distinct steps of RNA synthesis are not clearly characterized, either structurally or kinetically. This context of poorly defined mechanisms of RNA synthesis impedes precise evaluation of the inhibition potency of nucleotide analogues. The 2′-modified analogues are not obligate chain terminators. Therefore, a viral RNA terminated with such an analogue is expected to abort viral synthesis, hence the observed antiviral effect, but a 2′-modified nucleoside monophosphate incorporated into viral RNA and further elongated is also expected to exert an antiviral effect through the production of structurally altered progeny RNA genomes. Not known with precision either is the extent of discrimination of these analogues at the HCV polymerase active site during the initiation or elongation step. Clearly, a better understanding of the different steps of RNA synthesis is needed, as well as a molecular basis for the evaluation of inhibitor potency. This should aid the design of potent HCV inhibitors and the in vivo-in vitro correlation of their antiviral effect, as well as the anticipation of future potential resistance mechanisms that might develop upon sustained treatments. In this report, we contribute to the characterization of the initiation step of RNA synthesis. We have made use of 2′-O-methyl-GTP to dissect the effect of this GTP analogue on NS5B at the level of allosteric activation, inhibition of initiation, and elongation of RNA. We report discrimination values of this analogue relative to GTP during the initiation and elongation phases of RNA synthesis. We show that there is a marked discrimination of this analogue at the initiation of RNA synthesis, but not when RNA synthesis reaches the elongation phase. HCV 1b Polymerase Plasmid Constructions, Enzyme Preparation, and Reagents—Several reports (27Ranjith-Kumar C.T. Gutshall L. Sarisky R.T. Kao C.C. J. Mol. Biol. 2003; 330: 675-685Crossref PubMed Scopus (52) Google Scholar, 28Vo N.V. Tuler J.R. Lai M.M. Biochemistry. 2004; 43: 10579-10591Crossref PubMed Scopus (27) Google Scholar) mention that the authentic C-terminal part of NS5B is critical in the repression of primer-dependent synthesis in favor of initiation. Because we did not examine both RNA synthesis modes simultaneously, all NS5B proteins were truncated at their C terminus by 55 residues, and 6 histidine residues were added to the C terminus of each of the proteins to facilitate affinity purification. The resulting NS5B is called wild-type NS5B throughout this report, and it is unlikely that the former has a C terminus extending long enough to reach the active site (Fig. 2), as described for a 21-amino acid C terminus-truncated NS5B (29Adachi T. Ago H. Habuka N. Okuda K. Komatsu M. Ikeda S. Yatsunami K. Biochim. Biophys. Acta. 2002; 1601: 38-48Crossref PubMed Scopus (71) Google Scholar, 30Leveque V.J. Johnson R.B. Parsons S. Ren J. Xie C. Zhang F. Wang Q.M. J. Virol. 2003; 77: 9020-9028Crossref PubMed Scopus (48) Google Scholar). NS5B wild-type or mutant proteins were expressed from the pDest 14 vector (Invitrogen) in Escherichia coli BL21(DE3) cells (Novagen). Site-directed mutagenesis was made using the QuikChange site-directed mutagenesis kit according to the manufacturer's instruction (Stratagene). All constructions were verified by DNA sequencing. NS5B proteins were purified from bacteria grown at 37 °C in LB medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (17 μg/ml) and induced with 50 μm isopropyl 1-thio-β-d-galactopyranoside during 16–18 h at 17 °C. Bacteria were harvested by centrifugation, and recombinant RdRps were purified in sodium phosphate buffer through a Talon cobalt affinity column (Invitrogen) followed by SP-Sepharose column chromatography (Amersham Biosciences). Eluted proteins were concentrated to 2 mg/ml and stored at –20 °C. No attempts were made to titrate the active site concentration of NS5B preparations. Therefore, maximum velocity values (VM) related to total enzyme concentration are reported instead of kcat values throughout this report (see Tables I and II). RNA molecular weight markers were synthesized using T7 RNA polymerase and the appropriate template RNA. For this purpose, reactions were performed in T7 buffer (40 mm Tris-HCl, pH 7.5, 6 mm MgCl2, 2 mm spermidine, 10 mm dithiothreitol) at 30 °C for 10 min, using 10 ng of DNA oligonucleotide (5′-TTTTTTTTTTTTTTTTTTTTTTCCTATAGTGAGTCGTATTA-3′ or 5′-TTTTTTTTTTTTTTTTTTTCCCCCTATAGTGAGTCGTATTA-3′) template annealed to the T7 primer (5′-TAATACGACTCACTATAGGG-3′), and 10 μm [α-32P]GTP (1 μCi). Reactions were initiated by the addition of 1 μg of recombinant T7 RNA polymerase. The GMP marker was synthesized through hydrolysis of [α-32P]GTP by tobacco acid pyrophosphatase (Sigma) according to the manufacturer's instructions. RNA oligonucleotides were obtained from MWG-Biotech; homopolymeric cytosine template was obtained from Amersham Biosciences. DNA oligonucleotides were obtained from Invitrogen. For elongation experiments, RNA oligonucleotides were 5′-α-32P-labeled using T4 polynucleotide kinase (New England Biolabs). γ-32P-Labeled adenosine 5′-triphosphate (3000 Ci/mmol), α-32P-labeled guanosine 5′-triphosphate (3000 Ci/mmol), α-32P-labeled cytosine 5′-triphosphate (3000 Ci/mmol), and uniformly labeled [3H]GTP (5.20 Ci/mmol) were purchased from Amersham Biosciences. 2′-O-Methyl-GTP was purchased from Trilink, Inc.Table ISteady-state constants of wild-type, Δ-flap, and P495L NS5B polymerase for RNA templates during initiationWTΔ-flapP495LNucleotidesGTP2′-O-meGTPGTP2′-O-meGTPGTP2′-O-meGTPKm (μm)aKm and Vmax were calculated as described under “Experimental Procedures.”8.5 ± 0.8205 ± 2515 ± 1.770 ± 37.6 ± 0.6130 ± 25Vmax (s-1)bVmax is expressed in picomoles·s-1·pmol-1 enzyme.9.2 ± 0.2 × 10-31.4 ± 0.06 × 10-33.7 ± 0.09 × 10-31.1 ± 0.01 × 10-315.9 ± 0.3 × 10-31.6 ± 0.1 × 10-3Vmax/Km (s-1/μm)1 × 10-36.7 × 10-62.7 × 10-41.6 × 10-52 × 10-31.3 × 10-5Discrimination148-fold17-fold157-folda Km and Vmax were calculated as described under “Experimental Procedures.”b Vmax is expressed in picomoles·s-1·pmol-1 enzyme. Open table in a new tab Table IISteady-state constants of wild-type and Δ-flap NS5B polymerase on RNA templates during elongationWTΔ-flapNucleotidesGTP2′-O-meGTPGTP2′-O-meGTPKm (μm)47 ± 1436 ± 173.3 ± 0.918.3 ± 2.5Vmax (s-1)aVmax is expressed in percentage of GTP incorporated in hairpin template/s, normalized per μm enzyme as described under “Experimental Procedures.”5 ± 0.5 × 10-31.5 ± 0.5 × 10-30.2 ± 0.0050.2 ± 0.005Vmax/Km (s-1/μm)0.1 × 10-30.04 × 10-30.0550.01Discrimination2.3-fold5.5-folda Vmax is expressed in percentage of GTP incorporated in hairpin template/s, normalized per μm enzyme as described under “Experimental Procedures.” Open table in a new tab Inhibitory Concentration 50% (IC50) and Ki Determination—The 2′-O-methyl-GTP concentration leading to 50% inhibition of NS5B-mediated RNA synthesis was determined in RdRp buffer containing 100 nm homopolymeric cytosine RNA template, 0.1 mm [3H]GTP (0.5 μCi), and 2′-O-methyl-GTP (0, 0.1, 1, 2.5, 5, 7.5, 9, 10, 15, 20, and 25 μm). Reactions were initiated by the addition of 1 μm NS5B, incubated at 30 °C, and stopped after 3 min by spotting aliquots onto DE-81 paper discs (Whatman International Ltd.). Filter paper discs were washed three times for 10 min in 0.3 m ammonium formate, pH 8.0, washed two times in ethanol, and dried. The radioactivity bound to the filter was determined using liquid scintillation counting. IC50 was determined using the equation, % of activity =100/(1+(I)2/IC50)(Eq. 1) where I is the concentration of inhibitor. IC50 was determined from curve-fitting using KaleidaGraph (Synergy Software). Steady-state Incorporation of Nucleotide into Homopolymeric Templates—Polymerase activity was assayed by monitoring the incorporation of radiolabeled guanosine on 15-mers cytosine RNA oligonucleotide template. All indicated concentrations are final. The reaction was performed in RdRp buffer (50 mm HEPES, pH 8.0, 10 mm KCl, 5 mm MnCl2, 5 mm MgCl2, 10 mm dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.5% Igepal CA630) containing 10 or 100 μm GTP as indicated, 1 μCi of [α-32P]GTP, and 10 μm RNA oligonucleotide. Reactions were initiated by the addition of 1 μm purified NS5B and incubated at 30 °C. Aliquots were withdrawn over time from 10 s to 1 h. For inhibition analysis, increasing concentration of 2′-O-methyl-GTP (10, 50, 100, 200, or 400 μm) were added before the addition of NS5B, and the reaction was allowed to proceed for 30 min. Reaction products were separated using sequencing gel electrophoresis (20% acrylamide, 7 m urea in TTE buffer (89 mm Tris, 28 mm taurine, 0, 5 mm EDTA)) and quantitated using photo-stimulated plates and a FujiImager (Fuji). The formation of products was fitted with a burst equation, (P)=A⋅(1−exp(−(Vi⋅t))+kss⋅t)(Eq. 2) where A is the amplitude of the burst, Vi is the initial velocity of the reaction, and kss is the enzyme turn over rate. Determination of Vmax and Km—An RNA oligonucleotide corresponding to the 3′-end of the negative strand of the HCV genome (RNA H(–), 5′-UCGGGGGCUGGC-3′) was used to analyze the synthesis of the first phosphodiester bond. The RNA H(–) template (10 μm) was mixed in RdRp buffer with 1 μm NS5B. The reactions were started by addition of 100 μm [α-32P]CTP (1 μCi), GTP (1, 5, 10, 50, 100, and 500 μm), and incubated at 30 °C. Elongation was measured with a 5′ α-32P-labeled hairpin-RNA template (5′-UGACGGCCCGGAAAACCGGGCC-3′). The hairpin-RNA template (1 μm) was mixed with 1 μm NS5B in RdRp buffer before the addition of GTP (1, 5, 10, 50, 100, or 500 μm), and reactions were incubated at 30 °C for 1–30 min. Initiation or elongation reactions involving 2′-O-methyl-GTP were conducted using 2 μm enzyme to allow accurate measurement of low incorporation rates. All maximal velocities (VM values) were then normalized to 1 μm enzyme. Aliquots were withdrawn during the time course of the reaction, and the reactions were quenched with EDTA/formamide. Products were separated using sequencing gel electrophoresis and quantified using photo-stimulated plates and a FujiImager (Fuji). Product formation was fitted to a curve according to Equation 2. The dependence of Vi on NTP concentration is described by the hyperbolic equation, Vi=Vmax⋅(NTP)/(Km+(NTP))(Eq. 1) where Vmax and Km are the maximal velocity and the affinity constant of NTP incorporation by NS5B, respectively. Vmax and Km were determined from curve-fitting using KaleidaGraph (Synergy Software). GTP plays a special role during viral RNA synthesis by HCV NS5B. It is the initiating nucleotide of (+)RNA genome synthesis, it is incorporated throughout the genome during the elongation process, and its concentration is perhaps regulating theses steps through binding to an allosteric site (23Bressanelli S. Tomei L. Rey F.A. De Francesco R. J. Virol. 2002; 76: 3482-3492Crossref PubMed Scopus (337) Google Scholar, 31Tomei L. Altamura S. Bartholomew L. Biroccio A. Ceccacci A. Pacini L. Narjes F. Gennari N. Bisbocci M. Incitti I. Orsatti L. Harper S. Stansfield I. Rowley M. De Francesco R. Migliaccio G. J. Virol. 2003; 77: 13225-13231Crossref PubMed Scopus (193) Google Scholar, 32Ranjith-Kumar C.T. Kim Y.C. Gutshall L. Silverman C. Khandekar S. Sarisky R.T. Kao C.C. J. Virol. 2002; 76: 12513-12525Crossref PubMed Scopus (83) Google Scholar). Therefore, the mode of action of GTP analogues (such as ribavirin triphosphate) is expected to be complex. We have made use of a known GTP analogue inhibitor, 2′-O-methyl-GTP, to dissect kinetically the different mechanisms at work during initiation and elongation of RNA synthesis. The Target of 2′-O-Methyl-GTP and Its Inhibition Kinetics— When poly(rC) is used as a template of RNA synthesis, the appearance of poly(rG) RNA products is biphasic (Fig. 1A). The initial rate of RNA synthesis can be measured during the linear phase of the reaction, and the addition of increasing concentrations of 2′-O-methyl-GTP to the reaction decreases this rate of synthesis. The decrease of synthesis rate, i.e. inhibition, can be plotted as a function of 2′-O-methyl-GTP synthesis, giving a 50% inhibitory concentration (IC50) of 3.5 ± 0.2 μm (Fig. 1B). In an attempt to characterize further the mechanism of 2′-O-methyl-GTP inhibition, a traditional Henri-Michaelis-Menten analysis was performed, and the reciprocal Lineweaver-Burke plot is shown in Fig. 1C. The 2′-O-methyl-GTP analogue exhibits some features of a competitive inhibitor. However, above 200 μm GTP, all experimental curves merge with the uninhibited (I = 0) reaction curve, indicating that 2′-O-methyl-GTP ceases to exert its inhibitory activity. This non-linear behavior indicates that several phenomena are taking place in the same enzyme site and/or time, and thus, the kinetics of initiation and elongation of RNA were studied for between the GTP and its competing analogue. Kinetics of the Initiation Phase of RNA Synthesis Using GTP—The initiation phase of RNA synthesis is defined as the formation of the first phosphodiester bond between two nucleotides to form a ribo-dinucleotide primer. In the case of the HCV polymerase, structural studies have indicated that the active site is closed by the flap on one side and the fingers on the other side (Fig. 2). Thus, one can envision that nucleotides are polymerized up to a certain extent into RNA primers, then the active site has to open up to adopt a processive mode of elongative synthesis. These steps can be illustrated by a polymerization reaction analyzed with a gel assay. In this assay, an oligonucleotide of 15 consecutive cytidines (oligo(C)15) is used as a template to direct RNA synthesis using GTP as the sole nucleotide substrate. Fig. 3A shows a kinetic order of appearance of oligoG products. The size distribution of products generated using the oligoC15 template is identical to that obtained using poly(rC) (not shown). Abortive 6-mer products or less accumulate over time, whereas products >6-mer are rapidly and processively converted to 15-mer (template length, or G15) final products. In fact, a closer examination of the reaction products reveals that the overall reaction can be kinetically split into three phases (Fig. 3B). First, the very first phosphodiester bond formation from G1 to G2 occurs rapidly, and G2 products accumulate over time. Second, abortive products up to G6 are formed. Subsequently, a third phase occurs in which G6 products are elongated processively into full-length, run-off products. Fig. 3C shows the individual quantitation of reaction products. A band-product accumulating over time indicates a rate-limiting step for the subsequent phosphodiester bond formation. It is apparent that, out of the final G15 product, G2 and G5 accumulate more than G3, G4, and G6. Very similar patterns of product formation are observed in the presence of higher GTP concentrations (see “Supplementary Material,” Fig. S) as well as for a variety of templates (not shown). We conclude that, as opposed to the commonly reported hypothesis (33Lohmann V. Roos A. Korner F. Koch J.O. Bartenschlager R. Virology. 1998; 249: 108-118Crossref PubMed Scopus (128) Google Scholar, 34Lohmann V. Roos A. Korner F. Koch J.O. Bartenschlager R. J. Viral Hepat. 2000; 7: 167-174Crossref PubMed Scopus (66) Google Scholar), the true initiation reaction of formation of the G2 product is not rate-limiting, even at low GTP concentrations of 10 μm such as those used here. Instead, it is the next reaction from G2 to G3 which is rate-limiting together with another slow rate reaction from G6 to highly polymerized products. The formation of G2 or G3 to G6 and of >G6 products will be referred to as the initiation, transition, and elongation reaction, respectively. The initiation reaction was examined in greater kinetic details for GTP and 2′-O-methyl-GTP comparatively. Kinetic Analysis of the P1 to P2 Reaction, and Its Inhibition by 2′-O-Methyl-GTP—The kinetics of P2 product formation was examined using a gel assay as described above (Fig. 4A). To examine a single nucleotide incorporation and simplify the analysis, we made use of a short oligonucleotide template allowing the extension of a single GTP or 2′-O-methyl-GTP. As reported b" @default.
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