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

Bougie, Isabelle; Bisaillon, Martin

doi:10.1074/jbc.m400908200

Cited by 65 publications

(72 citation statements)

References 44 publications

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“…We therefore tested whether ribavirin-based analogs could functionally replace the 7-methylguanosine moiety of capped mRNA. Furthermore, viral capping enzymes can utilize RMP to form an RpppN cap on viral mRNAs (Bougie and Bisaillon 2004). We generated three versions of a firefly luciferase (F-luc) mRNA by in vitro transcription, incorporating the conventional m 7 GpppG structure, the RpppG structure, or GpppG (which does not bind eIF4E).…”

Section: Resultsmentioning

confidence: 99%

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

Westman¹,

Beeren²,

Grudzien³

et al. 2005

RNA

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The eukaryotic initiation factor eIF4E binds the mRNA 5 0 cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m 7 GTP and m 7 GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

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Section: Resultsmentioning

confidence: 99%

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

Westman¹,

Beeren²,

Grudzien³

et al. 2005

RNA

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“…The broad-spectrum antiviral ribavirin triphosphate, a nucleoside analog, has also been shown to directly interact with the guanylyltransferase of vaccinia virus, thereby inhibiting the guanylylation of RNA transcripts (Bougie and Bisaillon 2004). However, because of the high overall similarity between viral and cellular GTases, it was initially suggested that mechanism-based inhibitors might not be specific and show adverse side effects on human cells (Bougie and Bisaillon 2004). Nonetheless, mounting evidence suggests that subtle structural and mechanistic differences exist between the RNA viruses and cellular GTases.…”

Section: Discussionmentioning

confidence: 99%

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

Issur

Geiss²,

Bougie³

et al. 2009

RNA

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The 59-end of the flavivirus genome harbors a methylated m7 GpppA 29OMe cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 29-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.

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“…Within host cells, it alters GTP pool concentrations by inhibiting IMP dehydrogenase (IMPDH) (29,30). Other modes of action may include direct inhibition of the influenza virus RdRp (31,32) and interference with capping of viral RNA (33). Some data suggest that ribavirin affects inflammatory and T-cell responses in vivo (34)(35)(36).…”

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confidence: 99%

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

Pauly

Lauring

2015

J Virol

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Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. IMPORTANCEInfluenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. I nfluenza virus is a single-stranded, negative-sense RNA virus with a genome consisting of 8 segments (1). Like other RNA viruses, influenza virus replicates with extremely low fidelity. Its RNA-dependent RNA polymerase (RdRp) complex, which includes the viral proteins PB1, PB2, PA, and NP (2, 3), has a mutation rate of approximately 2.3 ϫ 10 Ϫ5 substitutions per nucleotide per cell ...

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

Cited by 65 publications

References 44 publications

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

Effective Lethal Mutagenesis of Influenza Virus by Three Nucleoside Analogs

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