Analysis of Flavivirus NS5 Methyltransferase Cap Binding

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 02/2009; 385(5):1643-54. DOI: 10.1016/j.jmb.2008.11.058
Source: PubMed

ABSTRACT The flavivirus 2'-O-nucleoside N-terminal RNA methyltransferase (MTase) enzyme is responsible for methylating the viral RNA cap structure. To increase our understanding of the mechanism of viral RNA cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (DEN) and yellow fever (YF) virus MTase enzymes. We solved an improved 2.1 A resolution crystal structure of DEN2 Mtase, new 1.5 A resolution crystal structures of the YF virus MTase domain in apo form, and a new 1.45 A structure in complex with guanosine triphosphate and RNA cap analog. Our structures clarify the previously reported DEN MTase structure, suggest novel protein-cap interactions, and provide a detailed view of guanine specificity. Furthermore, the structures of the DEN and YF proteins are essentially identical, indicating a large degree of structural conservation amongst the flavivirus MTases. Guanosine triphosphate analog competition assays and mutagenesis analysis, performed to analyze the biochemical characteristics of cap binding, determined that the major interaction points are (i) guanine ring via pi-pi stacking with Phe24, N1 hydrogen interaction with the Leu19 backbone carbonyl via a water bridge, and C2 amine interaction with Leu16 and Leu19 backbone carbonyls; (ii) ribose 2' hydroxyl interaction with Lys13 and Asn17; and (iii) alpha-phosphate interactions with Lys28 and Ser215. Based on our mutational and analog studies, the guanine ring and alpha-phosphate interactions provide most of the energy for cap binding, while the combination of the water bridge between the guanine N1 and Leu19 carbonyl and the hydrogen bonds between the C2 amine and Leu16/Leu19 carbonyl groups provide for specific guanine recognition. A detailed model of how the flavivirus MTase protein binds RNA cap structures is presented.

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Available from: Susan Keenan, Aug 17, 2015
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    • "391 NS3 DENV Hel and NS5 DENV MTase domains have been readily 392 crystallized as a monomeric free enzyme (Luo et al., 2008) and as 393 a dimeric complex with AdoMet (Lim et al., 2011), respectively, 394 yielding high-resolution X-ray diffraction data. Presence of AdoMet 395 co-purified with MTases from the bacterial lysates has already 396 been widely described (Benarroch et al., 2004; Egloff et al., 2002, 397 2007; Geiss et al., 2009; Lim et al., 2011; Yap et al., 2010). Since 398 one of the objectives of FBDD is to find new binding sites, we did 399 not attempt to exclude AdoMet from its binding pocket. "
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    ABSTRACT: Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0 Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2’-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180 μM to 9 mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites.
    Antiviral research 06/2014; 106(1). DOI:10.1016/j.antiviral.2014.03.013 · 3.94 Impact Factor
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    • "These results suggest that future effort on RNA-MTase cocrystal structure should use viral RNA, as opposed to various RNA cap analogues or nonviral RNAs. So far, cocrystal structures of MTase in complex with cap analogue have been obtained for several flaviviruses (Assenberg et al., 2007; Bollati et al., 2009; Egloff et al., 2007; Geiss et al., 2009); unfortunately, none of the structures captured the conformation for the N-7 or 2′-O catalysis. "
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    ABSTRACT: We report that dengue virus (DENV) methyltransferase sequentially methylates the guanine N-7 and ribose 2'-O positions of viral RNA cap (GpppA-->m(7)GpppA-->m(7)GpppAm). The order of two methylations is determined by the preference of 2'-O methylation for substrate m(7)GpppA-RNA to GpppA-RNA, and the 2'-O methylation is not absolutely dependent on the prior N-7 methylation. A mutation that completely abolished the 2'-O methylation attenuated DENV replication in cell culture, whereas another mutation that abolished both methylations was lethal for viral replication, suggesting that N-7 methylation is more important than 2'-O methylation in viral replication. The latter mutant with lethal replication could be rescued by trans complementation using a wild-type DENV replicon. Furthermore, we found that chimeric DENVs containing the West Nile virus methyltransferase, polymerase, or full-length NS5 were nonreplicative, but the replication defect could also be rescued through trans complementation using the wild-type DENV replicon.
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    • "Crystal structures of several flaviviral MTases in complex with cap analog dinucleotides have been determined (Assenberg et al., 2007; Egloff et al., 2007; Bollati et al., 2009c; Geiss et al., 2009). However, these structures provide little information regarding the RNA binding, since the second nucleotide following the GTP cap is distant from the methyl donor SAM, resulting in a conformation for the 2′-OH not suitable for MTase catalysis. "
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    ABSTRACT: Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5' terminal cap 1 structure (m(7)GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m(7)GpppA → m(7)GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.
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