2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase (original) (raw)
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Flavivirus RNA cap methyltransferase: structure, function, and inhibition
Frontiers in Biology, 2010
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.
Structure and Function of Flavivirus NS5 Methyltransferase
Journal of Virology, 2007
The plus-strand RNA genome of flavivirus contains a 5 terminal cap 1 structure (m 7 GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA 3 m 7 GpppA 3 m 7 GpppAm. The 2-O methylation can be uncoupled from the N-7 methylation, since m 7 GpppA-RNA can be readily methylated to m 7 GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2-O cap methylations require distinct buffer conditions and different side chains within the K 61 -D 146 -K 182 -E 218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations 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 N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.
Journal of Molecular Biology, 2007
The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase DV , is involved in two of four steps required for the formation of the viral mRNA cap 7Me GpppA 2′OMe , the guanine-N7 and the adenosine-2′O methylation. Its S-adenosyl-L-methionine (AdoMet) dependent 2′O-methyltransferase (MTase) activity has been shown on capped 7Me± GpppAC n RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 Å to 2.8 Å resolution of NS5MTase DV in complex with cap analogues and the coproduct of methylation S-adenosyl-L-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of 7Me GpppAC 4 and 7Me GpppAC 5 RNAs is inhibited in the presence of GTP, 7Me GTP and 7Me GpppA but not by ATP. This particular position of the cap is in accordance with the 2′O-methylation step. A model was generated of a ternary 2′O-methylation complex of NS5MTase DV , 7Me GpppA and AdoMet. RNAbinding increased when 7Me± GpppAGC n-1 starting with the consensus sequence GpppAG, was used instead of 7Me± GpppAC n . In the NS5MTase DV -GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5′ end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2′O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.
Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events
Journal of Virology, 2007
The 5 end of the flavivirus plus-sense RNA genome contains a type 1 cap (m 7 GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5 terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5 stem-loop structure; in contrast, 2-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5 viral RNA of 20 nucleotides. The cap analogues GpppA and m 7 GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp-and m 7 Gppp-terminated RNAs suggest that the 5 termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
Journal of Virology, 2008
Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA3m 7 GpppA-RNA3m 7 GpppAm-RNA, by using S-adenosyl-L-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAMbinding site donates methyl groups to both N7 and 2-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m 7 GpppA-RNA, after which the m 7 G moiety is repositioned to the GTP-binding pocket to register the 2-OH of the adenosine with SAM, generating m 7 GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.
Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain
Journal of Biological Chemistry, 2008
The Flavivirus NS5 protein possesses both (guanine-N7)methyltransferase and nucleoside-2-O methyltransferase activities required for sequential methylation of the cap structure present at the 5 end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-L-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)methyltransferase and nucleoside-2-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-L-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-L-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-L-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-L-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation. Cellular and many viral mRNAs contain a modified 5Ј-terminal guanosine "cap" structure covalently linked to the 5Ј end of
N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection
Cell host & microbe, 2016
The RNA modification N6-methyladenosine (m(6)A) post-transcriptionally regulates RNA function. The cellular machinery that controls m(6)A includes methyltransferases and demethylases that add or remove this modification, as well as m(6)A-binding YTHDF proteins that promote the translation or degradation of m(6)A-modified mRNA. We demonstrate that m(6)A modulates infection by hepatitis C virus (HCV). Depletion of m(6)A methyltransferases or an m(6)A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m(6)A sites across the HCV genome and determined that inactivating m(6)A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m(6)A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, ide...
Biochemical and genetic characterization of dengue virus methyltransferase
Virology, 2010
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 res...
Virology, 2008
West Nile virus methyltransferase catalyzes N7 and 2′-O methylations of the viral RNA cap (GpppA-RNA→m 7 GpppAm-RNA). The two methylation events are independent, as evidenced by efficient N7 methylation of GpppA-RNA→m 7 GpppA-RNA and GpppAm-RNA→m 7 GpppAm-RNA, and by the 2′-O methylation of GpppA-RNA→GpppAm-RNA and m 7 GpppA-RNA→m 7 GpppAm-RNA. However, the 2′-O methylation activity prefers substrate m 7 GpppA-RNA to GpppA-RNA, thereby determining the dominant methylation pathway as GpppA-RNA→m 7 GpppA-RNA→m 7 GpppAm-RNA. Mutant enzymes with different methylation defects can trans complement one another in vitro. Furthermore, sequential treatment of GpppA-RNA with distinct methyltransferase mutants generates fully methylated m 7 GpppAm-RNA, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.
Journal of Molecular Biology, 2009
The mRNA-capping process starts with the conversion of a 5V-triphosphate end into a 5V-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5V-5Vphosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-L-methionine to N7-guanine (cap 0) and to the ribose 2VOH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2VO-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-Lmethionine cofactor, S-adenosyl-L-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7Me GpppG, and N7Me GpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7methyltransferase and 2VO-methyltransferase activities.