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

Abstract

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...

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…The methyl transfer efficiency is similar (Fig. 3B), suggesting that both substrates can be methylated at the 2=-O position of the adenosine ribose, as already reported for DENV and WNV MTases (19,33). Additionally, the ZIKV MTase domain shows an optimal activity between pH 8 and 9 (Fig.…”

“…However, it is noteworthy that our biochemical assay might not be optimal to look for a possible interplay between the two domains as the RNA used by the MTase is not neo-synthesized by the RdRp. As observed for its close relatives DENV and WNV, the 2=-O-MTase activity can be detected using different short RNA substrates with no obvious sequence specificity (19,33). In contrast the N-7-MTase activity requires an RNA substrate forming a hairpin structure mimicking the SLA structure conserved among flaviviruses (7,20).…”

Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling

“…We first determined the impact of mutations affecting the ␣␤NLS on 2=O-MTase activity by measuring the transfer of a 3 Hradiolabeled methyl group contained in S-adenosylmethionine to a 5=-terminal fragment (corresponding to nucleotides 1 to 172) of the DENV-2 RNA genome (35). As shown in Fig.…”

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

“…Current literature highlights the development of effective nucleoside analogues tested in vitro that target RNA replication by inhibiting incorporation of nucleotides into the newly synthesized RNA strand (Cameron & Castro, 2001;Yin et al, 2009) or targeting the active site of the enzyme such as the S-adenosyl methionine (SAM) pocket of the MTase which is essential for methylation (Selisko et al, 2010). It has also been suggested that targeting interactions of nonstructural proteins in the replication complex is another approach to designing specific antivirals (Loregian et al, 2002).…”