The Broad Spectrum Antiviral Nucleoside Ribavirin as a Substrate for a Viral RNA Capping Enzyme
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m400908200
ISSN1083-351X
AutoresIsabelle Bougie, Martin Bisaillon,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoThe 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 noted previously, although the mechanism of action has not been explored (21Goswami B.B. Borek E. Sharma O.K. Fujitaki J. Smith R.A. Biochem. Biophys. Res. Comm. 1979; 89: 830-836Crossref PubMed Scopus (155) 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 RTP can be used as a substrate by the capping enzyme and can form a covalent RMP-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that RMP can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts, which possess ribavirin as the blocking nucleoside, are more stable than unblocked transcripts. The biological implications of these findings for the proposed ribavirin-mediated inhibition of RNA capping are discussed. Expression and Purification of the Vaccinia Virus D1 Protein (1-545)—A plasmid for the expression of the N-terminal segment of the vaccinia virus D1 protein was generated by PCR amplification of the sequence corresponding to residues 1-545 of the D1 coding region. The 5′- and 3′-primers harbored restriction sites for NheI and XhoI, respectively. Note that the 3′-primer was designed to introduce a translation stop at codon 546. The digested PCR product was inserted between the NheI and XhoI sites of the pET28a expression plasmid (Novagen). In this context, the resulting D1-(1-545) protein is fused in-frame with an N-terminal peptide containing six tandem histidine residues, and expression of the His-tagged protein is driven by a T7 RNA polymerase promoter. The resulting recombinant plasmid, pET-D1-(1-545), was transformed into Escherichia coli BL21(DE3). A 1-liter culture of E. coli BL21(DE3)/pET-D1-(1-545) was grown at 37 °C in Luria-Bertani medium containing 50 μg/ml kanamycin until the A600 reached 0.5. The culture was adjusted to 2% ethanol, and the incubation continued at 18 °C for 48 h. The cells were then harvested by centrifugation, and the pellet was stored at -80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria pellets were resuspended in 50 ml of lysis buffer A (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 10% sucrose), and cell lysis was achieved by the addition of lysozyme and Triton X-100 to final concentrations of 50 μg/ml and 0.1%, respectively. The lysates were sonicated to reduce viscosity, and any insoluble material was removed by centrifugation at 13,000 rpm for 45 min. The soluble extract was applied to a 5-ml column of nickel-nitrilotriacetic acid-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100. The column was washed with the same buffer and then eluted stepwise with buffer B (50 mm Tris-HCl pH 8, 0.1 m NaCl, and 10% glycerol) containing 50, 100, 200, 500, and 1000 mm imidazole. The polypeptide composition of the column fractions was monitored by SDS-PAGE. The recombinant D1-(1-545) protein was retained on the column and recovered in the 100 mm imidazole eluate. This fraction was applied to a 5-ml column of phosphocellulose that had been equilibrated in buffer C (50 mm Tris-HCl, pH 8, 50 mm NaCl, 2 mm dithiothreitol (DTT), 10% glycerol, and 0.05% Triton X-100). The column was washed with the same buffer and then eluted stepwise with buffer C containing 0.1, 0.2, 0.3, 0.4, 0.5, and 1 m NaCl. The recombinant protein was retained on the column and recovered predominantly in the 0.2 m NaCl fraction. The fraction was then dialyzed against buffer C that was supplemented with potassium pyrophosphate (1 mm) to ensure a homogenous nonguanylylated enzyme. The fraction was finally stored at -80 °C. Protein concentration was determined by the Bio-Rad dye binding method using bovine serum albumin as the standard. Assay for Enzyme-GMP Complex Formation—The assay was performed by incubating the enzyme with 10 μm [α-32P]GTP in a buffer containing 50 mm Tris-HCl, pH 8, 5 mm DTT, and 5 mm MgCl2 for 5 min at 37 °C. The reactions were stopped by the addition of EDTA to 10 mm and SDS to 1%. The reactions were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS. The radiolabeled proteins were visualized by autoradiography of the gel. The extent of covalent complex formation was quantitated by scanning the gel with a PhosphorImager (Amersham Biosciences). Assay for Enzyme-RMP Complex Formation—An assay similar to the one performed for the evaluation of the enzyme-GMP formation was used, except that [3H]RTP was utilized instead of [α-32P]GTP. Following electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS, the proteins were stained with Coomassie Blue dye. The corresponding bands were then excised from the gels and crushed, and the amount of radioactivity was determined with a liquid scintillation counter. Preparation of RNA Substrates—An RNA substrate of 84 nucleotides was synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. The RNA transcript was synthesized from the pBS-KSII+ plasmid (Stratagene) that had been linearized with HindIII. The RNA substrate was purified on a denaturing 20% polyacrylamide gel and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantitated by spectrophotometry. Alternatively, a radiolabeled RNA substrate was also synthesized by adding [α-32P]UTP to the transcription reaction. Transfer of RMP to RNA—Transfer of RMP to RNA was assayed by monitoring the transfer of [3H]RMP to an RNA transcript of 84 nucleotides (see above paragraph) in a buffer containing 50 mm Tris-HCl, pH 8, 2 mm MgCl2, 5 mm DTT, 2 μg of purified D1-(1-545) protein, and 1 mm [3H]RTP. The reaction was incubated at 37 °C for 30 min, and unincorporated [3H]RTP was removed by multiple rounds of ethanol precipitation. The RNA was extracted with phenol/chloroform and recovered by ethanol precipitation. Alternatively, a radiolabeled RNA substrate of 84 nucleotides was used (see above paragraph) and incubated with unlabeled RTP (1 mm) in a reaction mixture containing 50 mm Tris-HCl, pH 8, 2 mm MgCl2, 5 mm DTT, and 2 μg of purified D1-(1-545) protein. The RNA was extracted with phenol/chloroform, recovered by ethanol precipitation, and analyzed on a denaturing 20% polyacrylamide gel. The gel was then scanned with a PhosphorImager (Amersham Biosciences). In Vitro Stability of RNAs—The cellular protein extracts were prepared from a single 100-mm Petri dish culture of human embryonic kidney 293 (HEK293) cells. The cells were grown at 37 °C to 80% confluence prior to harvesting, and were then washed with PBS buffer (150 mm NaCl, 3 mm KCl, 10 mm Na2HPO4,and 2 mm KH2PO4, pH 7.4). The cells were treated with trypsin, centrifuged at 7,000 rpm for 1 min, resuspended in 4 ml of PBS buffer, and subjected to three freeze/thaw cycles. The protein extract was recovered after centrifugation at 13,000 rpm for 5 min to remove the cellular debris. The protein concentration of the extract was determined by the Bio-Rad dye binding method using bovine serum albumin as the standard. Protein extracts were added to an internally radiolabeled RNA species harboring an unblocked 5′-end or to RNAs possessing a 5′-end blocked with either guanosine, ribavirin, or the classical methylated guanosine cap. The reactions were incubated at 37 °C, and aliquots were recovered at various time points, extracted with phenol/chloroform, and precipitated with ethanol. The amount of radioactivity present in the ethanol-insoluble material was determined with a liquid scintillation counter. In Vitro Translation Assays—In vitro translation assays were performed in wheat germ cell-free extracts (Promega). The incubation (50 μl) was performed in the presence of 50 mm potassium acetate, 2.5 mCi of [35S]-methionine, and 10 μg of RNA species harboring an unblocked 5′-end, or to RNAs possessing a 5′-end blocked with either guanosine, ribavirin, or the classical methylated guanosine cap. The samples were incubated at 25 °C for 2 h, and aliquots (5 μl) were recovered at various time points and incubated in 1 m NaOH and 2% H2O2 at 37 °C for 10 min. The radiolabeled proteins were precipitated with 25% trichloroacetic acid and 2% casamino acids, and the acid-precipitable radioactivity was collected on glass fiber filters. The amount of incorporated radioactivity was evaluated with a liquid scintillation counter Formation of an RMP-Enzyme Covalent Intermediate—A His-tagged version of the vaccinia virus D1-(1-545) protein was expressed in bacteria and purified by sequential nickel-agarose and phosphocellulose chromatography steps (Fig. 1A). This N-terminal fragment of the D1 protein has been shown previously to be functionally equivalent to the full-sized capping enzyme with respect to triphosphatase and guanylyltransferase activities (22Higman M.A. Bourgeois N. Niles E.G. J. Biol. Chem. 1992; 267: 16430-16437Abstract Full Text PDF PubMed Google Scholar, 23Myette J. Niles E.G. J. Biol. Chem. 1996; 271: 11936-11944Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Shuman S. Morham S.G. J. Biol. Chem. 1990; 265: 11967-11972Abstract Full Text PDF PubMed Google Scholar, 25Yu L. Shuman S. J. Virol. 1996; 70: 6162-6188Crossref PubMed Google Scholar). The first step of the RNA guanylyltransferase reaction entails the nucleophilic attack of the α-phosphate of GTP by the enzyme and the subsequent formation of a covalent enzyme-GMP intermediate. The ability of the purified D1-(1-545) protein to form a covalent GMP-enzyme intermediate was detected by label transfer from [α-32P]GTP to the enzyme. A single SDS-stable GMP-enzyme complex that migrated as a 61-kDa species was detected following SDS-PAGE (Fig. 1B). Labeling of the enzyme was not detected in the absence of divalent cations (data not shown). We conclude that the D1-(1-545) protein is active in the formation of a protein-GMP covalent complex. Because the nucleoside analogue ribavirin is structurally related to guanosine (Fig. 2A), we intended to monitor the ability of RTP to serve as a substrate for the D1-(1-545) protein by measuring the covalent binding of [3H]RTP to the enzyme. Note that the tritium label is located on the triazole ring of RTP. The D1-(1-545) protein was incubated in the presence of magnesium and increasing concentrations of radiolabeled RTP, and the reaction products were submitted to SDS-PAGE. The ability of the D1-(1-545) protein to catalyze the formation of a covalent protein-RMP complex was demonstrated by label transfer from [3H]RTP. The proteins were stained with Coomassie Blue dye, and the corresponding bands were excised from the gel and crushed, and the radioactivity was evaluated by liquid scintillation counting. The yield of protein-RMP formation by the D1-(1-545) protein increased with RTP concentrations up to 300 μm and leveled off thereafter (Fig. 2B). Note that the amount of the protein-RMP complex formed during a 10-min incubation at 37 °C in the presence of 1 mm [3H]RTP was proportional to the amount of the added D1-(1-545) protein and that formation of the enzyme-RMP complex was dependent on the presence of a divalent ion (data not shown). These data demonstrate that RTP can serve as a substrate for the D1-(1-545) protein and form a stable covalent RMP-enzyme complex. A second assay was used to demonstrate the covalent transfer of RMP to the D1-(1-545) protein. In this assay, the enzyme was incubated in the presence of RTP and magnesium, and the polypeptide was analyzed by capillary electrophoresis. The appearance of a slower migrating protein species was observed repeatedly when the protein was incubated with RTP (Fig. 2C). We hypothesized that the slower migrating protein species corresponds to the enzyme with covalently bound ribavirin (RMP-enzyme). The addition of pyrophosphate to the typical RNA guanylyltransferase reaction has been shown to reverse the reaction, i.e. promote the release of GTP by reversal of the reaction (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, 26Shuman S. J. Biol. Chem. 1982; 257: 7237-7245Abstract Full Text PDF PubMed Google Scholar, 28Venkatesan S. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 340-344Crossref PubMed Scopus (42) Google Scholar). We therefore added pyrophosphate to the reaction to evaluate if the formation of the RMP-D1-(1-545) complex was reversible. Our results demonstrate that the slower migrating protein species was converted into the faster migrating species following incubation with pyrophosphate. These data clearly demonstrate that the D1-(1-545) protein can form a covalent RMP-enzyme complex and that the reaction is reversible. In the typical RNA guanylyltransferase reaction, the nucleophilic attack on the α-phosphate of GTP by the enzyme results in the formation of a covalent intermediate in which GMP is linked via a phosphoamide bond to a lysine residue of the enzyme (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, 29Niles E.G. Christen L. Virology. 1993; 193: 319-328Crossref PubMed Scopus (48) Google Scholar, 30Cong P. Shuman S. J. Biol. Chem. 1993; 268: 7256-7260Abstract Full Text PDF PubMed Google Scholar). To determine the nature of the enzyme-RMP linkage, the RMP-D1-(1-545) labeled complex was isolated by gel filtration and submitted to chemical treatment, and the products were analyzed by thin layer chromatography (Fig. 3B). The RMP-D1-(1-545) intermediate was resistant to NaOH treatment, but treatments with HCl or NH2OH resulted in the release of RMP, which is indicative of a phosphoamide linkage. As a control, the D1-(1-545) protein was labeled with GMP and analyzed in parallel to confirm the nature of the phosphoamide bond (Fig. 3A). Transfer of RMP to RNA—The D1-(1-545) protein clearly has the ability to form a covalent RMP-enzyme intermediate, but can it transfer RMP to an RNA substrate containing a 5′-diphosphate end? Because the D1-(1-545) protein harbors both the triphosphatase and guanylyltransferase active sites, the enzyme can hydrolyze the 5′-triphosphate end of RNAs to generate a 5′-diphosphate end that can subsequently serve as a substrate for capping. The ability of the enzyme to transfer RMP to an acceptor RNA molecule was tested by incubating the D1-(1-545) protein with [3H]RTP, an RNA substrate (84 nucleotides) containing a triphosphate 5′-end and magnesium. The products of the reaction were extracted with phenol/chloroform to remove the radiolabeled protein, and the RNA acceptor molecules were recovered by ethanol precipitation. Aliquots of the RNA samples were then digested with nuclease P1 and alkaline phosphatase and analyzed by polyethyleneimine-cellulose thin layer chromatography (Fig. 4B). The transfer of radiolabeled RMP to RNA was confirmed by demonstrating the release of a RpppG structure following digestion of the RNA samples with nuclease P1, with a simultaneous resistance to alkaline phosphatase treatment. Similar patterns were obtained when radiolabeled GTP was used in these experiments (Fig. 4A). We conclude that the D1-(1-545) protein can transfer the RMP moiety to an RNA acceptor molecule. A second assay was used to demonstrate the transfer of ribavirin to RNA. A 32P-radiolabeled RNA transcript (84 nucleotides) was synthesized by in vitro transcription and gel-purified after electrophoresis on a denaturing 20% polyacrylamide gel. The radiolabeled RNA was then incubated in the presence of the D1-(1-545) protein, ribavirin, and magnesium, and the reaction products were analyzed on a denaturing polyacrylamide gel. As can be seen in Fig. 4B, the addition of ribavirin, magnesium, and the D1-(1-545) protein to the radiolabeled RNA resulted in a slower migrating RNA species. We hypothesized that the slower migrating RNA species results from the addition of the ribavirin moiety to the RNA transcript. As observed in the typical RNA capping reaction, incubation of the reaction product with pyrophosphate drove the reaction in the opposite direction (18Shuman S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 187-191Crossref PubMed Scopus (116) Google Scholar, 26Shuman S. J. Biol. Chem. 1982; 257: 7237-7245Abstract Full Text PDF PubMed Google Scholar, 28Venkatesan S. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 340-344Crossref PubMed Scopus (42) Google Scholar). This demonstrates that the second step of the reaction, i.e. the D1-(1-545)-mediated transfer of RMP to RNA, is readily reversible. Stability of RNAs Blocked with Ribavirin—The presence of the cap structure found at the 5′-end of eukaryotic mRNAs has been shown to be involved in the stabilization of mRNAs by protecting the 5′-ends from exonucleolytic degradation (17Furuichi Y. LaFiandra A. Shatkin A.J. Nature. 1977; 265: 235-239Crossref PubMed Scopus (0) Google Scholar). The presence of a blocking, unmethylated capping guanosine residue at the 5′-end is sufficient for protection against degradation (31Furuichi Y. LaFiandra A. Shatkin A.J. Nature. 1977; 266: 235-239Crossref PubMed Scopus (348) Google Scholar). In an effort to evaluate the stability of RNAs capped with ribavirin, in vitro kinetic analyses were performed in the presence of total protein extracts isolated from human HEK293 cells. A radiolabeled RNA transcript (84 nucleotides) was synthesized by in vitro transcription and gel-purified after electrophoresis on a denaturing 20% polyacrylamide gel. The radiolabeled RNA was then blocked with either guanosine, ribavirin, or the classical methylated guanosine by incubating the transcript with the D1-(1-545) protein and magnesium. The various RNA species were then recovered by multiple rounds of ethanol precipitation. Note that the presence of a blocking nucleoside (guanosine or ribavirin) was confirmed by thin layer chromatography (data not shown). Radiolabeled RNAs were incubated at 37 °C in the presence of HEK293 total protein extracts, and aliquots were removed at various time points. As observed previously, our data indicate that RNA transcripts with an unblocked 5′-end are degraded more rapidly than RNAs blocked with GTP. The informative finding is that RNAs blocked with ribavirin are significantly more stable than unblocked RNAs (Fig. 5). In fact, 65% of the unblocked RNAs are degraded after 30 min in comparison to 20% for RNAs blocked with ribavirin. As shown in Fig. 5, the stability of RNAs harboring a RpppN 5′-end is similar to the stability of RNAs possessing a GpppN 5′-end. We conclude that the presence of a blocking ribavirin residue at the 5′-end of RNAs can protect them from exonucleolytic degradation. Translation of RNAs Blocked with Ribavirin—The presence of the 7-methyl group of the classical methylated guanosine 5′ cap has been shown previously to be critical for cap-dependent translation (32Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996Google Scholar). Various studies have demonstrated that the 7-methylguanosine interacts with the eukaryotic initiation factor 4E (eIF4E), a phylogenetically conserved subunit of the heterotrimeric eIF4F initiation complex, and establishes the foundation for the assembly of a functional translational initiation complex (33Niedzwiecka A. Marcotrigiano J. Stepinski J. Jankowska-Anyszka M. Wyslouch-Cieszynska A. Dadlez M. Gingras A.C. Mak P. Darzynkiewicz E. Sonenberg N. Burley S.K. Stolarski R. J. Mol. Biol. 2002; 319: 615-635Crossref PubMed Scopus (314) Google Scholar, 34Marcotrigiano M. Gingras A.C. Sonenberg N. Burley S.K. Cell. 1997; 89: 951-961Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 35Matsuo H. Li H. McGuire A.M. Fletcher C.M. Gingras A.C. Sonenberg N. Wagner G. Nat. Struct. Biol. 1997; 4: 717-724Crossref PubMed Scopus (325) Google Scholar). In an effort to investigate the relation between translation and stability of RNA transcripts blocked with ribavirin, in vitro translation studies were performed using wheat germ cell-free extracts (Fig. 5B). It is worth emphasizing that all attempts to methylate RNAs blocked with ribavirin by the vaccinia virus capping machinery were unsuccessful (data not shown). Our in vitro translation data indicate that only the RNA transcripts blocked with the classical m7GpppG cap structure were efficiently translated (Fig. 5B). Unblocked RNA transcripts, or RNAs blocked with either guanosine or ribavirin, were not efficiently translated. Overall, these results indicate that RNAs blocked with ribavirin are not efficie
Referência(s)