Energetics of RNA binding by the West Nile virus RNA triphosphatase

Benzaghou, Ines; Bougie, Isabelle; Picard-Jean, Frédéric; Bisaillon, Martin

doi:10.1016/j.febslet.2006.01.006

Cited by 15 publications

(13 citation statements)

References 53 publications

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“…This result is consistent with the exposure of the tryptophan residue to the polar solvent caused by the unfolding of the protein [41]. The fluorescence intensity also increased as a result of denaturation; increase in tryptophan fluorescence has been reported upon denaturation of proteins in urea [42], [43].…”

Section: Resultssupporting

confidence: 84%

Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

et al. 2013

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The hepatitis delta virus (HDV) is a small (∼1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.

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Section: Resultssupporting

confidence: 84%

Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

et al. 2013

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“…The binding of RNAs harboring modified RNA cap structures to the eIF4E protein was evaluated by fluorescence spectroscopy as described previously [23]. Briefly, excitation was performed at a wavelength of 290 nm fluorescence using a Hitachi F-2500 fluorescence spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

et al. 2013

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RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

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“…Expression and purification of the NS5 and NS3 protein from West Nile virus, Dengue virus, and Yellow fever virus was performed as described previously (Benzaghou et al 2006;Bougie and Bisaillon 2009;Geiss et al 2009). Briefly, recombinant fulllength WNV NS5 protein (strain NY1999, amino acids 1-905), truncated WNV NS3 protein (strain NY99, amino acids 168-618), and the NS5 MTase domains from DV (strain 16681, amino acids 1-267), YFV (strain 17D, amino acids 1-268), and WNV (strain NY1999, amino acids 1-268) were expressed from inducible T7 expression plasmids that contain a carboxy-terminal 6-histidine tag.…”

Section: Expression and Purification Of The Flavivirus Ns5 Proteinmentioning

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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Energetics of RNA binding by the West Nile virus RNA triphosphatase

Cited by 15 publications

References 53 publications

Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

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

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