A Structural Basis for the Inhibition of the NS5 Dengue Virus mRNA 2′-O-Methyltransferase Domain by Ribavirin 5′-Triphosphate

Abstract

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5′-triphosphate inhibits the activity of the dengue virus 2′-O-methyltransferase NS5 domain (NS5MTaseDV). Along with several other guanosine 5′-triphosphate analogues such as acyclovir, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5′-triphosphate competes with GTP to bind to NS5MTaseDV. A structural view of the binding of ribavirin 5′-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTaseDV, ribavirin 5′-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 Å. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5′-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses. Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5′-triphosphate inhibits the activity of the dengue virus 2′-O-methyltransferase NS5 domain (NS5MTaseDV). Along with several other guanosine 5′-triphosphate analogues such as acyclovir, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5′-triphosphate competes with GTP to bind to NS5MTaseDV. A structural view of the binding of ribavirin 5′-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTaseDV, ribavirin 5′-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 Å. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5′-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses. The guanosine analogue ribavirin is a broad spectrum antiviral agent discovered almost 30 years ago (1Sidwell R.W. Huffman J.H. Khare G.P. Allen L.B. Witkowski J.T. Robins R.K. Science. 1972; 177: 705-706Crossref PubMed Scopus (854) Google Scholar). Since its discovery, many mechanisms of action have been proposed (reviewed in Refs. 2Patterson J.L. Fernandez-Larsson R. Rev. Infect. Dis. 1990; 12: 1139-1146Crossref PubMed Scopus (235) Google Scholar and 3Hong Z. Cameron C.E. Prog. Drug Res. 2002; 59: 41-69Crossref PubMed Scopus (75) Google Scholar). Like most nucleoside analogues, ribavirin is phosphorylated by cellular kinases at its 5′-position upon entry into the cell. Ribavirin 5′-monophosphate is a potent inhibitor of the cellular enzyme inosine 5′-monophosphate dehydrogenase. This inhibition results in the depletion of the intracellular guanosine nucleotide pool, which feeds capping and polymerase enzymes from both viral and cellular origin. Consequently, the depressed guanosine nucleotide pool may exert an indirect antiviral effect, since viral enzymes would not compete advantageously for guanosine nucleotides with cellular enzymes. In addition, ribavirin nucleotides may have a viral target, such as RNA polymerization and RNA capping or induce lethal mutagenesis of viral genomes (4Crotty S. Maag D. Arnold J.J. Zhong W. Lau J.Y. Hong Z. Andino R. Cameron C.E. Nat. Med. 2000; 6: 1375-1379Crossref PubMed Scopus (697) Google Scholar), accounting for the observed antiviral effect. Both direct and indirect mechanisms may thus contribute to the ribavirin mode of action. To date, ribavirin nucleotides have been crystallized with two cellular enzymes, namely inosine 5′-monophosphate dehydrogenase (5Prosise G.L. Wu J.Z. Luecke H. J. Biol. Chem. 2002; 277: 50654-50659Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and nucleoside diphosphate kinase (NDPK (6Gallois-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)) but not with any viral enzyme or protein. The genus Flavivirus comprises important human pathogens such as West Nile, dengue, and yellow fever viruses, which are moderately sensitive to ribavirin (7Neyts J. Meerbach A. McKenna P. De Clercq E. Antiviral Res. 1996; 30: 125-132Crossref PubMed Scopus (95) Google Scholar, 8Jordan I. Briese T. Fischer N. Lau J.Y. Lipkin W.I. J. Infect. Dis. 2000; 182: 1214-1217Crossref PubMed Scopus (162) Google Scholar, 9Crance J.M. Scaramozzino N. Jouan A. Garin D. Antiviral Res. 2003; 58: 73-79Crossref PubMed Scopus (239) Google Scholar). These mosquito-borne viruses are currently expanding their distribution throughout the world. The introduction of West Nile virus in North America may be an important milestone in the history of this virus, as exemplified by outbreaks in the New York area (10Anderson J.F. Andreadis T.G. Vossbrinck C.R. Tirrell S. Wakem E.M. French R.A. Garmendia A.E. Van Kruiningen H.J. Science. 1999; 286: 2331-2333Crossref PubMed Scopus (289) Google Scholar) followed by the gradual spread to 47 of the 49 continental states of the United States of America (www.cdc.gov/ncidod/dvbid/westnile/index.htm). The Camargue area in France has re-witnessed West Nile viral infection of horses after 40 years, and the first human cases were reported in the French Riviera in October 2003. Likewise, dengue virus, an agent responsible for hemorrhagic fever, infects more than 50 million persons annually with an increasing incidence in tropical areas around the world. The single-stranded RNA genome of flaviviruses is of positive polarity, and is capped with a cap 1 structure Me7GpppA2′OMe (11Chambers T.J. Hahn C.S. Galler R. Rice C.M. Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1589) Google Scholar). The N-terminal domain of the dengue virus polymerase NS5 is a 2′-O-methyltransferase that is active on RNA cap structures (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). This enzyme, referred to as NS5MTaseDV, is able to bind a GTP molecule that may mimic the RNA cap structure prior to methylation. Thus, it was of interest to determine whether guanosine analogues could bind to the GTP binding site of NS5MTaseDV. If so, guanosine analogues may act as potential competitive inhibitors of RNA cap binding and aid in the rational design of inhibitors directed against flaviviruses. In this report, we present biochemical evidence that ribavirin 5′-triphosphate (RTP) 1The abbreviations used are: RTP, ribavirin 5′-triphosphate; RMP, ribavirin 5′-monophosphate; SAHC, S-adenosyl-l-homocysteine; PDB, Protein Data Bank; EICAR, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide; HPLC, high pressure liquid chromatography; GDPMP, β-γ-methylene GTP.1The abbreviations used are: RTP, ribavirin 5′-triphosphate; RMP, ribavirin 5′-monophosphate; SAHC, S-adenosyl-l-homocysteine; PDB, Protein Data Bank; EICAR, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide; HPLC, high pressure liquid chromatography; GDPMP, β-γ-methylene GTP. inhibits the 2′-O-methyltransferase activity of dengue virus NS5MTaseDV. We also show that a series of RTP analogues, as well as EICAR 5′-triphosphate (EICAR-TP) and acyclovir 5′-triphosphate (acyclovir-TP), compete with GTP for binding to NS5MTaseDv. In addition to show that RTP and GTP share a common binding site on NS5MTaseDV, the crystal structure of the RTP-NS5MTaseDV complex reveals a unique mode of binding for the ribavirin pseudobase that is consistent with a lack of discrimination of this antiviral molecule relative to GTP. Enzymes and Reagents—The purification and crystallization of the NS5 capping domain of the dengue virus RNA-dependent RNA polymerase has been described (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). Synthesis and purification of RTP, ribavirin nucleotide analogues, and acyclovir 5′-TP to homogeneity, as determined by 1H, 13C, and 31P NMR and HPLC, have been described (6Gallois-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). EICAR 5′-TP was a kind gift from P. Herdewijn (Leuven, Belgium). All other nonradioactive and 32P-labeled nucleotides were of HPLC grade and purchased from Amersham Biosciences. Inhibition of RNA 2′-O-Methyltransferase Activity by RTP—The methyltransferase assay was performed in 40 mm Tris-HCl, pH 7.1. 100 μmS-adenosylmethionine with 10 μCi of Ado[methyl-3H]Met (74 Ci/mmol; Amersham Biosciences) were incubated with 2 μg of purified NS5MTaseDV, 35 μl of RNA substrate, and various concentrations of ribavirin 5′-triphosphate (50, 100, 250, 300, 500 and 750 μm) in a 50-μl reaction mix (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). The RNA substrate mix was produced using purified T7 DNA primase in conjunction with the synthetic DNA oligonucleotide T10CTG5 (10 μm), 300 μm CTP, and 200 μm cap analogue to obtain capped AC5 (GpppAC5 and and m7GpppAC5) as described (13Matsuo H. Moriguchi T. Tagaki T. Kusakabe T. Buratowski S. Sekine M. Kyogoku Y. Wagner G. J. Am. Chem. Soc. 2000; 122: 2417-2421Crossref Scopus (22) Google Scholar). The methyl-transfer reaction was incubated at 30 °C and monitored from 30 min to 3 h. 8-μl aliquots were spotted onto DEAE-81 filter paper (Whatman) and washed with 20 mm sodium formate, pH 8.0 to remove any remaining S-adenosylmethionine. The experiment without ribavirin 5′-triphosphate served as a reference. Radioactively labeled RNA was quantified by liquid scintillation counting. Determination of RTP Dissociation Constant for NS5MTaseDV—The dissociation constant, Kd, was determined using 58 μm [32P]GTP and increasing concentrations of RTP (0, 100, 200, 500, 800, and 1000 μm) incubated with 2 μg of NS5MTaseDV. The bound radioactivity was quantitated after SDS-PAGE using photostimulatable plates and a FujiImager. Data were fit to a hyperbolic function from which the Kd was determined. GTP-binding Inhibition by RTP and Its Analogues—Two μg of NS5MTaseDV was incubated with RTP or RTP analogues and with [32P]GTP (50 μm, 10% volume of [32P]GTP) in 50 mm Tris, pH 7.6, 5 mm dithiothreitol, and 5 mm MgCl2. The reaction mixture was irradiated with UV light for 3 min using a UV lamp (40 watts, at 254 nm) at a distance of 12 mm, boiled for 5 min, and subjected to denaturing gel electrophoresis. The gel was stained using Coomassie Blue dye, and radioactive products were visualized using photostimulatable plates and a FujiImager. Structure Determination and Refinement—Crystals of NS5MTaseDV were grown by vapor diffusion as described (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar) and further soaked for 3 h in 2 mm RTP. A 2.6-Å data set was collected at 100 K using a charge-coupled device detector (ADSC Q4) at ID14-2 beamline, ESRF, Grenoble, France. Data were processed using DENZO (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar), and intensities were merged with SCALA (15P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar). Since the unit cell dimensions of the crystal were slightly different from those of the apo-form, the structure was solved by the molecular replacement program AmoRe, whereas polypeptide chain of the apo-form served as a model (PDB code 1L9K). A random set comprising 5% of the data was omitted from refinement for Rfree calculation (16Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3860) Google Scholar). A round of simulated annealing refinement, as implemented using crystallography CNS software, was performed (17Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). in the of the apo-form, was observed for the first and the 30 for one molecule of S-adenosyl-l-homocysteine and one molecule of ribavirin 5′-monophosphate were into the and The model was further and using the A. C. Scholar) and CNS NMR this which were present in the and were are in I. have been in the Protein Data code and refinement cell of of unique All data were used with are set of the not used in the All data were used with Rfree are set of the not used in the in a Inhibition of the RNA 2′-O-Methyltransferase Activity of NS5MTaseDV NS5MTaseDV domain is able to bind GTP (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). In addition, the crystal structure of a ternary complex consisting of NS5MTaseDV, SAHC, and GTP has been described (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). In this GTP an RNA Since NS5MTaseDV as an RNA 2′-O-methyltransferase (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar), we whether RTP could have a direct on this Since ribavirin is a guanosine analogue and a inhibitor of (7Neyts J. Meerbach A. McKenna P. De Clercq E. Antiviral Res. 1996; 30: 125-132Crossref PubMed Scopus (95) Google Scholar, 8Jordan I. Briese T. Fischer N. Lau J.Y. Lipkin W.I. J. Infect. Dis. 2000; 182: 1214-1217Crossref PubMed Scopus (162) Google Scholar, 9Crance J.M. Scaramozzino N. Jouan A. Garin D. Antiviral Res. 2003; 58: 73-79Crossref PubMed Scopus (239) Google Scholar, J.L. Antiviral Res. PubMed Scopus Google Scholar, P. Van A. C. H. De Clercq E. J. PubMed Scopus Google Scholar), the NS5MTaseDV domain may be a for RTP. In the RNA 2′-O-methyltransferase a capped RNA is incubated with NS5MTaseDV, the and various concentrations of RTP. The RNA is monitored using a filter paper binding assay (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). the results of such an inhibition RTP inhibits the RNA the the to a inhibition is 20 μm, to the that RTP is an inhibitor of the RNA 2′-O-methyltransferase activity in the NS5MTaseDV of the to the NS5MTaseDV crystal structure of NS5MTaseDV (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar) that this domain has two binding one for and the other for which could for the binding of RTP and inhibition of the RNA 2′-O-methyltransferase Since RTP is a GTP a GTP-binding experiment was used to the RTP binding the is incubated with a of GTP and increasing concentrations of RTP, GTP binding is This that RTP GTP from NS5MTaseDV. the relative inhibition is against RTP an Kd of μm is RTP a NS5MTaseDV binding to that of GTP 58 μm (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google We the binding of other analogues to NS5MTaseDV. 5′-triphosphate is a guanosine analogue with an the and EICAR 5′-triphosphate is a ribavirin Both analogues bound to NS5MTaseDV with Kd of and μm, that RTP has the of the guanosine analogues and that the of acyclovir 5′-TP is important for binding than a such as that of EICAR to assay other RTP analogues on their (6Gallois-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). A series of was performed to determine whether any of analogues would bind to NS5MTaseDV than RTP. 2 the of such in which and were used to compete for GTP of analogues a for NS5MTaseDV than RTP. almost as as RTP, whereas with the relative We that RTP and GTP compete for the NS5MTaseDV binding and the structural basis for this was further using for the of RTP to the NS5MTaseDV and for Its crystal of NS5MTaseDV was soaked for 3 h in 2 mm RTP the described (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). The structure was solved using molecular replacement and that an RTP molecule was bound to the enzyme, and the RTP molecule was at the GTP binding for the GTP analogue GDPMP, the and of RTP were present but not well in the and were not The and the of RTP with NS5MTaseDV in a to the GTP analogue A and are the or pseudobase and the of both The structural of ribavirin with guanosine from the of both the and and in the analogue of with the and the at the the interactions of the with the (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). The complex of NS5MTaseDV with the GTP analogue or RTP not in any of the protein. the of both it that ribavirin the by the of the of by a around an to the and the of the a the of ribavirin is with the of the and is in a the of RTP not with any and the a to the of and as the of Thus, RTP the atomic as and this is in with the that RTP and GTP We that RTP is a inhibitor of the RNA 2′-O-methyltransferase activity by the NS5MTaseDV In light of and structural RTP as a GTP or RNA cap to the RNA cap binding site of the NS5MTaseDV and RNA cap In this we have show that RTP inhibits the RNA 2′-O-methyltransferase activity of dengue virus NS5MTaseDV We further the binding site of RTP binding to the cap binding site using GTP The RTP binding site was at the atomic using crystallography at 2.6 The structure is the first of a viral enzyme to a ribavirin cellular structures have been solved in complex with ribavirin the of nucleoside diphosphate kinase from (PDB code (6Gallois-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)) and the dehydrogenase from (PDB code (5Prosise G.L. Wu J.Z. Luecke H. J. Biol. Chem. 2002; 277: 50654-50659Abstract Full Text Full Text PDF PubMed Scopus (35) Google In the the binding of RTP is the and of RTP with the but the ribavirin is an which could be by any and The atomic are different in the complex of with as the ribavirin pseudobase with the In the ternary complex with A (PDB code structural as the in a binding site and the interactions of ribavirin with the are not a of the relative of and in complex with inosine 5′-monophosphate dehydrogenase reveals that ribavirin with to is observed in the This is of the of both with the of ribavirin could have been by the in the whereas with the or the could have been by the of is not whether ribavirin NS5MTaseDV in has been that ribavirin the of several with the viral (7Neyts J. Meerbach A. McKenna P. De Clercq E. Antiviral Res. 1996; 30: 125-132Crossref PubMed Scopus (95) Google Scholar, 8Jordan I. Briese T. Fischer N. Lau J.Y. Lipkin W.I. J. Infect. Dis. 2000; 182: 1214-1217Crossref PubMed Scopus (162) Google Scholar, 9Crance J.M. Scaramozzino N. Jouan A. Garin D. Antiviral Res. 2003; 58: 73-79Crossref PubMed Scopus (239) Google Scholar, J.L. Antiviral Res. PubMed Scopus Google Scholar, P. Van A. C. H. De Clercq E. J. PubMed Scopus Google Scholar). In the of dengue and West Nile, and yellow fever viruses, the reported are and μm (7Neyts J. Meerbach A. McKenna P. De Clercq E. Antiviral Res. 1996; 30: 125-132Crossref PubMed Scopus (95) Google Scholar, 9Crance J.M. Scaramozzino N. Jouan A. Garin D. Antiviral Res. 2003; 58: 73-79Crossref PubMed Scopus (239) Google Scholar, P. Van A. C. H. De Clercq E. J. PubMed Scopus Google Scholar). The inhibition observed in this for RTP μm) are in with ribavirin the intracellular GTP it the of RTP against GTP for NS5MTaseDV the intracellular GTP concentrations in various cell have been and to be mm in A. T. DNA W. H. New Scholar). of with 20 μm ribavirin the GTP to a to of that of the around μm J. A. L. C. I. A. Van A. Herdewijn P. De Clercq E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This is the as that of the for NS5MTaseDV μm (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google This that with ribavirin which ribavirin concentrations of μm in 30: PubMed Scopus Google Scholar), the GTP is not to NS5MTaseDV, the that the binding of RTP to NS5MTaseDV may be to the ribavirin This may also be to other RTP has been reported to be an inhibitor of the of virus, as well as to to RNA cap in (reviewed in M. G. 1997; PubMed Scopus Google Scholar). To date, is evidence that ribavirin viral enzymes other than RNA as in the of Z. Cameron C.E. Prog. Drug Res. 2002; 59: 41-69Crossref PubMed Scopus (75) Google Scholar, 2003; PubMed Scopus Google Scholar). The structural data a model for the inhibition of the RNA 2′-O-methyltransferase activity by RTP. RTP may compete with viral RNA cap structures to bind the and RNA cap methylation. or not RTP inhibition the of results have in of drug of the Protein Data that of GTP with the in and has been reported that of NS5MTaseDV (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). The of cellular enzymes to at two of the at or (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (482) Google Scholar). or cap two (PDB and bind GTP using a in this in addition to In since RTP and GTP have and at 1 and but not RTP could bind to GTP-binding using 1 and To the of is of such binding in the Protein Data a of human origin. The of an antiviral for ribavirin may that ribavirin viral this 2 of or both viral and cellular GTP-binding 1 and and the of but has a more on viral the observed the series of RTP analogues has been used to a RNA-dependent RNA polymerase (6Gallois-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). not have an RNA capping but their RNA-dependent RNA polymerase domain is to that of flaviviruses. We that the most important for are not the virus polymerase and NS5MTaseDV. virus the is (6Gallois-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), whereas present results that the is most important in the of NS5MTaseDV. the most important is also the for the RNA it to design a that would both the RNA polymerase and the enzyme of flaviviruses are in the binding of RTP to NS5MTaseDV. A of an chain RTP relative to GTP in to provide potential drug Since RNA capping is for various viruses A. C. Scholar), the mechanism for inhibition of RNA capping may in for the antiviral activity of ribavirin against but it not the inhibition of viral results would provide a basis for rational drug design against human pathogens of viral of which the emerging flaviviruses are a We and for of the