An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization

Abstract

contributed equally to this work Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of¯avi-viruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A Ê resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2¢-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP-and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTPbinding site, suggest that the latter is a speci®c capbinding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging¯avi-viruses. Keywords: crystal structure/¯avivirus/GTP/RNA capping/RNA polymerase IntroductionThe cap is a unique structure found at the 5¢-end of viral and cellular eukaryotic mRNA (Bisaillon and Lemay, 1997;Furuichi and Shatkin, 2000). It is critical for both mRNA stability and binding to the ribosome during translation. mRNA capping is a co-transcriptional modi®cation resulting from a series of three chemical reactions (Shuman, 2001). The 5¢-triphosphate of the mRNA is ®rst converted to a diphosphate by an RNA triphosphatase. The second reaction is a transfer of a guanosine monophosphate (GMP) moiety from GTP to the 5¢-diphosphate RNA by a guanylyltransferase to yield G 5 ¢-ppp 5 ¢-N. In a third reaction utilizing S-adenosyl-L-methionine (AdoMet) as the methyl donor, the transferred guanosine moiety is methylated by a (guanine-N7)-methyltransferase (N7MTase) to yield 7Me G 5 ¢-ppp 5 ¢-N (cap 0 structure). A second methylation reaction catalysed by a (nucleoside-2¢-O-)-methyltransferase (2¢OMTase) occurs on the ®rst nucleotide 3¢ to the triphosphate bridge to yield 7Me G 5 ¢-ppp 5 ¢-N Me (cap 1 structure). Adjacent nucleotides 3¢ to the ®rst one can also be 2¢-O-methylated to various extents. The order of the methyltransfer reactions is variable, and in some viruses GTP is methylated at its N7 position before being transferred to the RNA 5¢-end (Ahola and Kaariainen, 1995;Furuichi and Shatkin, 2000).Few enzymes involved in the RNA capping pathway have been structurally characterized. The crystal structures of two cellular RNA triphosphatases from yeast and mouse have been determined at 2.0 and 1.6 A Ê resolution, respectively. These structures provide a mechanistic insight into the ®rst reaction in the RNA capping pathway catalysed by prototypic enzymes of the metal-dependent and -indep...

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

“…We have demonstrated previously that the recombinant MTase domain of NS5MTase DV transfers a methyl group from co-factor AdoMet to the 29O position of the first nucleotide of purified capped oligonucleotides 7Me GpppAC n and GpppAC n (Egloff et al, 2002;Peyrane et al, 2007). In a non-radioactive test, reverse-phase HPLC was used to analyse reaction mixtures (Fig.…”

“…Interestingly, both N7-MTase and 29O-MTase activities are located in the same protein, the N-terminal domain of protein NS5 (Egloff et al, 2002;Ray et al, 2006;Zhou et al, 2007), which also bears the RNA-dependent RNA polymerase function in the C-terminal domain (Selisko et al, 2006;Yap et al, 2007). Many flavivirus MTase domains have been characterized structurally (Assenberg et al, 2007;Bollati et al, 2009;Egloff et al, 2002;Geiss et al, 2009;Mastrangelo et al, 2007;Zhou et al, 2007). They adopt the common fold of AdoMet-dependent class 1 MTases (Schubert et al, 2003).…”

“…The tested NS5MTase DV potential inhibitor compounds were from Sigma-Aldrich: AdoHcy, 3-deaza-adenosine, SIBA, sinefungin Expression and purification of NS5MTase DV . Recombinant NS5MTase DV corresponding to residues 1-296 of NS5 was expressed and purified as described previously (Egloff et al, 2002).RNA synthesis, purification and binding assays. Capped and non-capped RNA ( 7Me GpppAC 5 and GpppAC 5 , pppAC 5 ) were synthesized in vitro using bacteriophage T7 DNA primase and purified by HPLC as described by Peyrane et al (2007).…”

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…Although the exact membrane topology of NS2A is yet to be determined, NS2A of OHFV has been predicted to span the membrane of the endoplasmic reticulum five times by several transmembrane domain prediction programs (TMHMM (Krogh et al, 2001), and TMpred (Hofmann and Stoffel, 1993)). The Leu46 residue in NS2A is located in the conserved hydrophobic NS5 is the largest (104kDa) of the flavivirus proteins, and three functional domains have been identified in NS5: a S-adenosylmethionine methyltransferase-like domain in the N-terminal region (Egloff et al, 2002;Koonin, 1993;Ray et al, 2006), a centrally located nuclear localization sequence (NLS) (Forwood et al, 1999;Kapoor et al, 1995), and an RNA-dependent RNA polymerase (RdRp) domain in the C-terminal region (Bartholomeusz and Wright, 1993;Koonin, 1991). The Asp836 residue in NS5 is located in the RdRp domain.…”

Struktur Proteomik Virus Dengue Dan Manfaatnya Sebagai Target Antivirus

“…Hal tersebut dikarenakan fungsi penting protein tersebut, yaitu sebagai metiltransferase (MTase; residu 1-296) pada N-terminal, serta sebagai RNA-dependent RNA polymerase (RdRp; residu 320-900) pada C-terminal. 20 Protein NS5 MTase merupakan salah satu protein virus yang banyak diteliti sebagai target antivirus. Untuk proses 5'-capping pada RNA virus, dibutuhkan empat macam aktivitas enzim, salah satunya MTase.…”