The Broad Spectrum Antiviral Nucleoside Ribavirin as a Substrate for a Viral RNA Capping Enzyme

The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavirin-mediated inhibition of capping are discussed. The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavirin-mediated inhibition of capping are discussed. Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses (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 (842) Google Scholar, 2De Clercq E. Adv. Virus Res. 1993; 42: 1-55Crossref PubMed Scopus (168) Google Scholar). Ribavirin is a synthetic purine nucleoside analogue with a structure closely related to guanosine (3Prusiner P. Sundaralingam M. Nat. New Biol. 1973; 244: 116-118Crossref PubMed Scopus (74) Google Scholar). Once inside the cells, ribavirin is phosphorylated by cellular kinases, with ribavirin triphosphate (RTP) 1The abbreviations used are: RTP, ribavirin triphosphate; RMP, ribavirin monophosphate; DTT, dithiothreitol; HEK293, human embryonic kidney 293. 1The abbreviations used are: RTP, ribavirin triphosphate; RMP, ribavirin monophosphate; DTT, dithiothreitol; HEK293, human embryonic kidney 293. as the major intracellular metabolite (4Miller J.P. Kigwana L.J. Streeter D.G. Robins R.K. Simon L.N. Roboz J. Ann. N. Y. Acad. Sci. 1977; 284: 211-229Crossref PubMed Scopus (74) Google Scholar, 5Page T. Connor J.D. Int. J. Biochem. 1990; 22: 379-383Crossref PubMed Scopus (126) Google Scholar). A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin. For instance, ribavirin monophosphate (RMP) has been shown to inhibit the host cell inosine monophosphate dehydrogenase, an enzyme involved in the de novo synthesis of GTP (6Streeter 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 (449) Google Scholar, 7Muller W.E. Maidhof A. Taschner H. Zahn R.K. Biochem. Pharmacol. 1977; 26: 1071-1075Crossref PubMed Scopus (68) Google Scholar). Because GTP is required for the transcription of viral genomes and replication of RNA viruses, it has been assumed that the decrease in the cytosolic concentration of GTP could affect the multiplication of viruses. Ribavirin also modulates the host immune system by engendering a bias toward helper T-cell type 1 cytokine response (8Fang S.H. Hwang L.H. Chen D.S. Chiang B.L. J. Hepatol. 2000; 33: 791-798Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Zuckerman E. Zuckerman T. Sahar D. Streichman S. Attias D. Sabo E. Yeshurun D. Rowe J.M. Blood. 2001; 97: 1555-1559Crossref PubMed Scopus (135) Google Scholar). This would ultimately lead to an enhanced immune response against viral infections. Ribavirin has also been shown to have an inhibitory effect on viral polymerases by competitively inhibiting the binding of the nucleotides (10Eriksson B. Helgstrand E. Johansson N.G. Larsson A. Misiorny A. Noren J.O. Philipson L. Stenberg K. Stening G. Stridh S. Oberg B. Antimicrob. Agents Chemother. 1977; 11: 946-951Crossref PubMed Scopus (196) Google Scholar, 11Lau J.Y. Tam R.C. Liang T.J. Hong Z. Hepatology. 2002; 35: 1002-1009Crossref PubMed Scopus (308) Google Scholar, 12Cameron C.E. Castro C. Curr. Opin. Infect. Dis. 2001; 14: 757-764Crossref PubMed Scopus (108) Google Scholar). More recently, elegant studies demonstrated that ribavirin can actually be used by viral polymerases and incorporated into viral RNA with the potential to base pair with UMP and CMP, leading to ribavirin-mediated mutagenesis of viral genomes (13Crotty 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 (684) Google Scholar, 14Maag D. Castro C. Hong Z. Cameron C.E. J. Biol. Chem. 2001; 276: 46094-46098Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 15Crotty S. Cameron C.E. Andino R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6895-6900Crossref PubMed Scopus (689) Google Scholar). This would ultimately drive the viruses beyond a critical mutation rate and lead to an overall reduced fitness of the viral populations. Other mechanisms of action have been proposed for ribavirin but have not been fully explored, including the possibility that ribavirin may have a negative effect on the capping of viral RNA transcripts (12Cameron C.E. Castro C. Curr. Opin. Infect. Dis. 2001; 14: 757-764Crossref PubMed Scopus (108) Google Scholar). The 5′-end of most eukaryotic mRNAs and many viral mRNAs harbors a m7GpppN cap structure that plays a critical role in the translation and stability of mRNAs (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (710) Google Scholar, 17Furuichi Y. LaFiandra A. Shatkin A.J. Nature. 1977; 265: 235-239Crossref PubMed Scopus (0) Google Scholar). The first step in the synthesis of the cap structure involves the hydrolysis of the RNA 5′-triphosphate end of the nascent RNA by an RNA triphosphatase to form a diphosphate end. An RNA guanylyltransferase enzyme then catalyzes a two-step reaction in which it initially utilizes GTP as a substrate to form a covalent GMP-enzyme intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the GpppN structure (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar). The guanosine residue is finally methylated by an RNA methyltransferase to form the typical m7GpppN cap structure. Support for the possibility that ribavirin might inhibit the capping of viral RNA transcripts comes from the finding that mutations in the Sindbis virus genome that confer resistance to ribavirin map to the RNA guanylyltransferase coding region of the genome (19Scheidel L.M. Stollar V. Virology. 1991; 181: 490-499Crossref PubMed Scopus (93) Google Scholar, 20Scheidel L.M. Durbin R.K. Stollar V. Virology. 1987; 158: 1-7Crossref PubMed Scopus (63) Google Scholar). Furthermore, the inhibition of vaccinia virus RNA cap synthesis by ribavirin has been the of action has not been E. J. Biochem. Res. PubMed Scopus Google Scholar). In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that can be used as a substrate by the capping enzyme and can form a covalent intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA which possess ribavirin as the blocking are more stable than unblocked transcripts. The biological implications of these findings for the proposed ribavirin-mediated inhibition of RNA capping are discussed. and of the Virus for the of the of the vaccinia virus by of the to of the coding The and for and that the to a translation The the and of the In the is with an and of the is by a RNA The into A of E. in the The to and the for The then by and the in of A and and cell by the of and to of and The to and by for The to a of that been with A The with the and then with and and The of the by The on the and in the This to a of that been in and The with the and then with and 1 The on the and in the The then against that with to a enzyme. 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J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar, L. S. J. 70: PubMed Google Scholar). The first step of the RNA guanylyltransferase reaction the of the of GTP by the enzyme and the of a covalent intermediate. The ability of the purified to form a covalent GMP-enzyme intermediate by from to the enzyme. A GMP-enzyme that as a of the enzyme not in the of not We that the is in the of a covalent Because the nucleoside analogue ribavirin is related to guanosine we to the ability of to serve as a substrate for the by the covalent binding of to the enzyme. that the is on the of The in the of and of RTP, and the reaction to The ability of the to the of a covalent demonstrated by from The with and the from the and and the by The of by the with to and that the of the during a in the of 1 to the of the and that of the on the of a not data demonstrate that can serve as a substrate for the and form a stable covalent A second used to demonstrate the covalent of to the In the enzyme in the of and and the by The of a the with We that the to the enzyme with ribavirin The of to the typical RNA guanylyltransferase reaction has been shown to the the of GTP by of the reaction (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We to the reaction to the of the Our demonstrate that the into the with data demonstrate that the can form a covalent and that the reaction is In the typical RNA guanylyltransferase the on the of GTP by the enzyme in the of a covalent intermediate in which GMP is a to a residue of the enzyme (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, L. Virology. 1993; PubMed Scopus Google Scholar, P. S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). the of the the by and to and the by The intermediate to but with in the of RMP, which is of a a the with GMP and in to the of the of to has the ability to form a covalent but can it to an RNA substrate a Because the harbors the triphosphatase and guanylyltransferase the enzyme can the 5′-triphosphate end of to a end that can serve as a substrate for The ability of the enzyme to to an RNA by the with an RNA substrate a triphosphate 5′-end and The of the reaction with to the and the RNA by of the RNA then with and and by The of to RNA by the of a structure of the RNA with with a resistance to GTP used in these We that the can the moiety to an RNA A second used to demonstrate the of ribavirin to A RNA transcript by in vitro transcription and on a The RNA then in the of the ribavirin, and and the reaction on a can be in the of ribavirin, and the to the RNA in a RNA We that the RNA from the of the ribavirin moiety to the RNA in the typical RNA capping of the reaction with the reaction in the (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This that the second step of the the of to is of with of the cap structure the 5′-end of eukaryotic mRNAs has been shown to be involved in the of mRNAs by the from Y. LaFiandra A. Shatkin A.J. Nature. 1977; 265: 235-239Crossref PubMed Scopus (0) Google Scholar). The of a capping guanosine residue the 5′-end is for against Y. LaFiandra A. Shatkin A.J. Nature. 1977; 235-239Crossref PubMed Scopus Google Scholar). In an to the stability of with ribavirin, in vitro in the of from human A RNA transcript by in vitro transcription and on a The RNA then blocked with ribavirin, the classical methylated guanosine by the transcript with the and The RNA then by of that the of a blocking nucleoside by not in the of and our data indicate that RNA transcripts with an unblocked 5′-end are more than blocked with The finding is that blocked with ribavirin are more stable than unblocked In of the unblocked are 30 in to for blocked with ribavirin. shown in the stability of a RpppN 5′-end is to the stability of a GpppN We that the of a blocking ribavirin residue the 5′-end of can from of with of the of the classical methylated guanosine cap has been shown previously to be critical for translation N. Scholar). studies have demonstrated that the with the eukaryotic a of the and the for the of a A. J. J. M. A. M. P. E. N. R. J. Biol. 2002; PubMed Scopus Google Scholar, M. N. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. N. G. Nat. Biol. PubMed Scopus Google Scholar). In an to the translation and stability of RNA transcripts blocked with ribavirin, in vitro translation studies is that to blocked with ribavirin by the vaccinia virus capping not Our in vitro translation data indicate that the RNA transcripts blocked with the classical cap structure translated RNA blocked with guanosine ribavirin, not these indicate that blocked with ribavirin are not by the and not serve as for for an to the of as a substrate for the capping we the of and GTP in The in the of and The as and The of as a of GTP concentration and The that a concentration of of the also However, the of that concentration of that of Furthermore, a concentration of of required to that GTP is more than in the of the covalent intermediate. This also by in which the ability of to inhibit the RNA guanylyltransferase activity by the purified investigated of to the RNA guanylyltransferase and and the effect on the of the covalent shown to inhibit the RNA guanylyltransferase activity by the by and by The findings that inhibition present more than GTP the possibility that might inhibit the guanylyltransferase by as a to with GTP for the in the broad spectrum antiviral ribavirin from studies the of the nucleoside analogue in the of and ability to inhibit the replication of major a major potential as a biological G. S. S. E. Med. J. Google Scholar, M. Res. PubMed Scopus Google Scholar). of action is ribavirin has been shown to the replication of a number of DNA and RNA viruses (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 (842) Google Scholar, 2De Clercq E. Adv. Virus Res. 1993; 42: 1-55Crossref PubMed Scopus (168) Google Scholar). has previously been that ribavirin could have a negative effect on the capping of viral RNA possibility has not been fully (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (710) Google Scholar, L.M. Stollar V. Virology. 1991; 181: 490-499Crossref PubMed Scopus (93) Google Scholar, 20Scheidel L.M. Durbin R.K. Stollar V. Virology. 1987; 158: 1-7Crossref PubMed Scopus (63) Google Scholar, E. J. Biochem. Res. PubMed Scopus Google Scholar). Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The of to the of ribavirin as a substrate for the vaccinia virus RNA capping enzyme. Our indicate that the vaccinia virus enzyme has the ability to form a covalent intermediate and the moiety to an RNA are to the typical RNA capping reaction in which GTP is used as the capping RNA are of the covalent which also DNA and RNA S. B. PubMed Scopus Google Scholar). the of a structure of an binding and a C. V. S. Cell. 11: Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A.J. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J.Y. C. J. J. J. 2000; PubMed Scopus Google Scholar, M. V. S. Cell. 2000; Full Text Full Text PDF Scopus Google Scholar). of the virus RNA capping enzyme the and the GTP substrate K. A.J. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, have been shown to during substrate binding and reaction K. A.J. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The and the of ribavirin to of the and the of However, an of ribavirin is the of the and the in A for the of the vaccinia virus RNA capping enzyme for GTP is that the has an not in RTP, that with the enzyme. the of GTP has been shown to be involved in a with a of the binding of the enzyme K. A.J. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the of GTP also with and of the K. A.J. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of these in reaction has been but our study that which the and of is a more substrate than for capping studies indicate that ribavirin can be used as a substrate by the RNA polymerases of the and the virus (13Crotty 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 (684) Google Scholar, 14Maag D. Castro C. Hong Z. Cameron C.E. J. Biol. Chem. 2001; 276: 46094-46098Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 15Crotty S. Cameron C.E. Andino R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6895-6900Crossref PubMed Scopus (689) Google Scholar). can as a substrate in a it However, the of to be than that of nucleotides (13Crotty 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 (684) Google Scholar, 14Maag D. Castro C. Hong Z. Cameron C.E. J. Biol. Chem. 2001; 276: 46094-46098Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 15Crotty S. Cameron C.E. Andino R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6895-6900Crossref PubMed Scopus (689) Google Scholar). catalytic is to and inhibitory on RNA The for of have not been investigated but are reminiscent of the that is in our study as a substrate for the vaccinia virus RNA capping enzyme. mechanisms by which ribavirin could inhibit the capping of viral RNA transcripts have been previously (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (710) Google Scholar). Ribavirin has been shown to a of the cellular GTP the inhibition of inosine monophosphate an enzyme required for the de novo of GTP (6Streeter 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 (449) Google Scholar, 7Muller W.E. Maidhof A. Taschner H. Zahn R.K. Biochem. Pharmacol. 1977; 26: 1071-1075Crossref PubMed Scopus (68) Google Scholar). A decrease in GTP could have a negative effect on the capping of viral for comes from studies with the Sindbis virus and the virus that that the viruses in with ribavirin the of GTP to the capping of viral (19Scheidel L.M. Stollar V. Virology. 1991; 181: 490-499Crossref PubMed Scopus (93) Google Scholar, 20Scheidel L.M. Durbin R.K. Stollar V. Virology. 1987; 158: 1-7Crossref PubMed Scopus (63) Google Scholar, T. J. PubMed Google Scholar). However, evidence that the antiviral effect of ribavirin is not the of the intracellular GTP For instance, inosine monophosphate as are not viral (13Crotty 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 (684) Google Scholar). The second by which ribavirin could inhibit the capping of viral is by binding directly to the of viral capping the binding of Ribavirin a base to a studies that the structure of ribavirin closely that of guanosine (3Prusiner P. Sundaralingam M. Nat. New Biol. 1973; 244: 116-118Crossref PubMed Scopus (74) Google Scholar). The of the is of it ribavirin to our studies indicate that can be used as a substrate by the vaccinia virus capping GTP a more substrate for the enzyme. we not the possibility that may have an of in the of GTP are the inhibition of inosine monophosphate Ribavirin is a that can utilize including a negative effect on the capping of viral RNA transcripts that can overall antiviral The activity of ribavirin is also by the that viruses that not as the virus and the are also to ribavirin (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (710) Google Scholar). have been used previously to inhibit the in of the cap structure by the cellular the inhibition of G. 1993; PubMed Scopus Google Scholar). In our study for the first that a nucleoside analogue can be directly by a viral capping enzyme and transferred to an RNA However, biochemical assays that ribavirin could not be methylated by the vaccinia virus RNA capping not The of on the blocking ribavirin residue to be as by our in vitro translation assays that indicate that RNA transcripts blocked with ribavirin are not by the cellular of action for antiviral Because of the of the cap structure for translation and viral involved in the synthesis of the cap structure are for the of antiviral with ability to interact with the RNA capping has the potential to serve as a for the of more In a number of ribavirin have been in of the these and RNA capping also on the of the RNA capping is the biological of the present studies that the of ribavirin is in by ribavirin can on the virus and cell used M. Res. PubMed Scopus Google Scholar, T. J. PubMed Google Scholar). However, for the that ribavirin in with GTP has been in viral For the antiviral effect of ribavirin can be by the of but not by nucleotides in with the virus and the virus (6Streeter 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 (449) Google Scholar, T. J. PubMed Google Scholar). The by which the antiviral ribavirin in activity a negative effect on capping is to However, our study that the vaccinia virus capping enzyme can interact with the antiviral ribavirin and it as a in studies in the of ribavirin indicate the antiviral ribavirin as a substrate cellular GTP are We Simon for on the We also and for the and for of vaccinia virus

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