West Nile Virus Methyltransferase Catalyzes Two Methylations of the Viral RNA Cap through a Substrate-Repositioning Mechanism

Abstract

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.Eukaryotic mRNAs contain a 5Ј cap structure that is essential for RNA splicing, export, stability, and translation (12). In general, RNA capping consists of four steps. (i) The 5Ј-triphosphate end of the nascent RNA transcript is hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase; (ii) the GMP moiety of GTP is transferred to the 5Ј diphosphate of RNA by an RNA guanylyltransferase; (iii) the N7 position of guanine is methylated by an RNA guanine-methyltransferase (N7 MTase), yielding a cap 0 structure (GpppN); and (iv) the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase, so as to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (12). S-Adenosyl-L-methionine (SAM) is the methyl donor for both the N7 and 2Ј-O methylations, generating Sadenosyl-L-homocysteine (SAH) as a by-product. Because host mRNA capping occurs in the nucleus, viruses replicating in the cytoplasm encode their own unique machineries for RNA capping. For example, the plus-strand RNA alphaviruses methylate GTP prior to the transfer of m 7 GMP to the 5Ј diphosphate of the RNA (2). The minus-strand RNA vesicular stomatitis virus (VSV) transfers GDP, rather than GMP, to the 5Ј monophosphate of the RNA (24). The differences between the host and viral cap formation processes could potentially be used for development of antiviral therapy.The genus Flavivirus contains a number of significant human pathogens, including the four serotypes of dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus, West Nile virus (WNV), and tick-borne encephalitis virus (6). Among those, DENV alone was estimated to cause 50 million human cases annually (38). Flaviviruses replicate in the cytoplasm. The viral ge...

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

“…Currently, the test is being further optimized to use lower enzyme and substrate concentrations. Interestingly, it was shown that sinefungin inhibits WNV replication with an EC 50 of 23 mM and a therapeutic index of 167 (Dong et al, 2008a). Thus, although it has also been reported to inhibit mammalian MTases (Chrebet et al, 2005;Osborne et al, 2007), sinefungin or its analogues might show specificity towards selected viral AdoMet-dependent MTases depending on 29O-methyltransferase activity of dengue virus NS5 subtle differences in their AdoMet-binding sites.…”

“…We tested various AdoMet analogues (see Methods), among them molecules with proven viral mRNA cap MTase inhibition capacity, such as the reaction product AdoHcy (Pugh & Borchardt, 1982) and sinefungin (Dong et al, 2008a;Li et al, 2007;Pugh et al, 1978), but also molecules inhibiting other AdoMet-dependent MTases, such as 59-S-isobutylthio-59-deoxyadenosine (SIBA), 3-deaza-adenosine (Kloor et al, 2004) and 59-deoxy-59-methylthio-adenosine (MTA) (Woodcock et al, 1983), or simply analogues of adenosine (29,39,59-tri-O-acetyl-adenosine) and AdoMet. We also used three GTP analogues: broad-spectrum antiviral ribavirin and its triphosphate, as well as 5-ethynyl-1-b-Dribofuranosylimidazole-4-carboxamide) (EICAR) triphosphate.…”

“…With respect to the flavivirus MTase as a potential target of antivirals, reverse genetic experiments using replicon and genome-length RNA have shown that mutations of N7-and/or 29O-MTase catalytic residues block or attenuate replication (Ray et al, 2006;Zhou et al, 2007). Accordingly, the AdoMet analogue sinefungin has been shown to inhibit both methylations and to suppress WNV replication in cell culture (Dong et al, 2008a). The crucial role of methylation of the cap structure for virus replication has also been targeted in other viral systems testing analogues of GTP or AdoMet as competitive inhibitors against MTases of viruses from the genera Alphavirus, Orthopoxvirus, Avulavirus (Paramyxoviridae) and Vesiculovirus (Rhabdoviridae) (Lampio et al, 1999;Li et al, 2007;Pugh et al, 1978).…”

Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs

“…It has been generally assumed that like cellular RNAs, many viral RNAs have a 5′ m7G cap, and that viruses either use the cellular capping machinery (e.g., viruses that replicate in the nucleus) or encode their own capping enzymes (e.g., viruses that replicate in the cytoplasm) (15,16). Interestingly, there is a surprisingly diverse range of cap modifications; for instance, Mimivirus, a large DNA virus of amoeba, encodes an RNA cap guanine-N2 methyltransferase that hypermethylates the viral RNA cap (17); influenza virus "snatches" caps from cellular mRNAs (18,19); West Nile fever virus has two methyl additions on its RNA cap (20); Sindbis virus produces mRNAs that have dimethylguanosine and trimethylguanosine caps [hypermethylated caps/m 2,2,7 G caps (21)]; and Semliki forest virus late mRNAs also have hypermethylated caps (22). On the other hand, poliovirus, encephalomyelitis virus, foot and mouth disease virus, and Calici virus are examples of viral RNAs lacking 5′ caps (11,23), and there is limited in vitro evidence that HIV-1 RNAs are capped (24,25).…”

Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases

“…In the case of chlorella virus, the RTPase, GTase and (Hodel et al, 1996); in West Nile virus (WNV), NS5 protein has only one MTase domain catalysing both N 7 -MTase and 29-O-MTase reactions through a substrate-repositioning mechanism (Dong et al, 2008;Ray et al, 2006;Zhou et al, 2007), whereas in the case of BTV, protein VP4 is not only responsible for the GTase and RTPase activities (Martinez-Costas et al, 1998), but also has MTase activity .…”