Flaviviral methyltransferase/RNA interaction: Structural basis for enzyme inhibition

Milani, Mario; Mastrangelo, Eloise; Bollati, Michela; Selisko, Barbara; Decroly, Étienne; Bouvet, Mickaël; Canard, Bruno; Bolognesi, Martino

doi:10.1016/j.antiviral.2009.03.001

Cited by 62 publications

(66 citation statements)

References 28 publications

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“…Interestingly, mutational analysis of NS5MTase WNV within the viral genome has shown that the 29O-MTase activity is important for WNV reproduction (Zhou et al, 2007). Our 29O-MTase assay will be useful for screening and characterizing potential inhibitors, as already demonstrated by the identification of two flavivirus MTase inhibitors (Luzhkov et al, 2007;Milani et al, 2009). …”

Section: Resultsmentioning

confidence: 97%

“…NS5MTase DV did not show any N7-MTase activity on the substrate GpppAC n . The N7-MTase activity of NS5MTase DV has been demonstrated on long (190 nt) specific RNA substrates corresponding to the 59 end of flavivirus genomes (Dong et al, 2007;Milani et al, 2009). Using 39 end deletions of a WNV substrate, it was shown that the N7-MTase activity of NS5MTase WNV required a 74 nt substrate including the 59 stem-loop A present in all flavivirus genomes (Dong et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

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Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

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The flavivirus RNA genome contains a conserved cap-1 structure, 7Me GpppA 29OMe G, at the 59 end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)-and the (nucleoside-29O)-MTases (29O-MTase), reside in a single domain of nonstructural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 29O-MTase activity of NS5MTase of dengue virus (NS5MTase DV ) using purified, short, capped RNA substrates ( 7Me GpppAC n or GpppAC n ). NS5MTase DV methylated both types of substrate exclusively at the 29O position. The efficiency of 29O-methylation did not depend on the methylation of the N7 position. Using 7Me GpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTase DV 29O activity and substrate binding increased before reaching a plateau at n55. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7Me GpppAC 5 and the co-substrate S-adenosyl-Lmethionine (AdoMet) were determined (0.39 and 3.26 mM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 29O-MTase activity of NS5MTase DV showed a low turnover of 3.25¾10 "4 s "1 . Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase DV , which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC 50 0.34 mM) and sinefungin (IC 50 0.63 mM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays. INTRODUCTIONMost eukaryotic and viral mRNAs are modified by the addition of a cap structure at the 59 end. The mRNA cap consists of an N7-methylguanosine linked to the first transcribed nucleotide by a 59-59 triphosphate bridge (cap-0 structure, 7Me GpppN) (Shuman, 2001). Methylation of the 29O positions of the ribose of the first or the second transcribed nucleotide leads to a cap-1 ( 7Me GpppN 29OMe ) or cap-2 ( 7Me GpppN 29OMe N 29OMe ) structure, respectively. The cap structure stabilizes mRNA and plays a vital role in its translation by controlling mRNA splicing, its transport across the nuclear membrane and its recognition by the eIF4E translation factor (Furuichi & Shatkin, 2000). In eukaryotic cells, the acquisition of the cap-0 structure is a co-transcriptional event. RNA capping implies three steps in which the 59 triphosphate end of RNA is hydrolysed to a 59 diphosphate by an RNA triphosphatase (RTPase), then capped with GMP by a guanylyltransferase (GTase) and finally methylated at the N7 position of the guanine by an S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) (Gu & Lima, 2005). In contrast to mRNAs of lower eukaryotes, which bear a cap-0 structure, the mRNAs of higher eukaryotes acquire a cap-1 structure via the action of a (nucleoside-29O)-MTase (29O-MTase) (Langberg & Moss, 1981). Compared with N7 methylation, this mRNA methylation step seems to be less important for binding, tr...

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

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“…Enzyme-based screenings or structure-based drug design have identified DENV, WNV, and YFV MTase inhibitors targeting either the SAM/SAH (S-adenosyl-Lhomocysteine, the coproduct of the methylation) binding pocket (26)(27)(28), the cap binding pocket (29), or allosteric sites (30,31). As ZIKV is closely related, we initiated a comparative study of ZIKV MTase with other Flavivirus MTases with the aim of subsequently developing original inhibitors or repurposing inhibitors targeting DENV MTase for antiviral research on ZIKV.…”

mentioning

confidence: 99%

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

Coutard

Barral

Lichière

et al. 2017

J Virol

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124

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The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2=-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2=-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-L-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the ␣X ␣-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns,and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.KEYWORDS methyltransferase, RNA processing, Zika virus, flavivirus, protein structure-function

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“…ATA was also shown to inhibit the DENV and WNV MTases, with a proposed binding site linked to the KDKE RNA-binding motif. 20 We therefore tested the ability of ATA to disrupt FL-dT10-YFV MTase complexes. Results, shown in Figure 3, indicate that ATA disrupted the binding of FL-dT10 to YFV MTase with an EC 50 = 1.54 ± 0.16 µM, which is close to the IC 50 for ATA inhibition of human DNA MTases DNMT1, 0.68 µM, and DNMT3a, 1.4 µM.…”

Section: Ata a Small-molecule Inhibitor Of Protein Nucleic Acid Intementioning

confidence: 99%

“…18 The inhibitor of nucleic acid binding, aurintricarboxylic acid (ATA), was first identified as a potent inhibitor of DENV and WNV MTases by means of in silico screening. 20 Here we describe a simple high-throughput assay for discovery of inhibitors of flavivirus MTases in which fluoresceinlabeled dT10 (FL-dT10) bound to the YFV MTase is used as a surrogate for the vRNA for the discovery of a new class of flaviviral inhibitors, compounds with vRNA displacement activity. This high-throughput assay works without the immediate need for radioactivity-based enzymatic reactions and is suitable for miniaturization.…”

Section: Introductionmentioning

confidence: 99%

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

Falk

Weisblum

2014

SLAS Discovery

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RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5′ end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a K d = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC 50 for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5′-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z′ of 0.83 and was successfully used to screen a library of known bioactives.

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Flaviviral methyltransferase/RNA interaction: Structural basis for enzyme inhibition

Cited by 62 publications

References 28 publications

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

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