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

The flavivirus RNA genome contains a conserved cap-1 structure, 7MeGpppA2′OMeG, at the 5′ end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2′O)-MTases (2′O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2′O-MTase activity of NS5MTase of dengue virus (NS5MTaseDV) using purified, short, capped RNA substrates (7MeGpppAC n or GpppAC n ). NS5MTaseDV methylated both types of substrate exclusively at the 2′O position. The efficiency of 2′O-methylation did not depend on the methylation of the N7 position. Using 7MeGpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTaseDV 2′O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7MeGpppAC5 and the co-substrate S-adenosyl-l-methionine (AdoMet) were determined (0.39 and 3.26 μM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2′O-MTase activity of NS5MTaseDV showed a low turnover of 3.25×10−4 s−1. Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTaseDV, which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC50 0.34 μM) and sinefungin (IC50 0.63 μM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.