HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (Papers in Press[PDF]) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Selisko, B. Articles by Decroly, E. PubMed PubMed Citation Articles by Selisko, B. Articles by Decroly, E. Agricola Articles by Selisko, B. Articles by Decroly, E.

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

AFMB, CNRS-Université de la Méditerranée

¹ E-mail: barbara.selisko{at}afmb.univ-mrs.fr

The Flavivirus RNA genome contains a conserved cap-1 structure, ^7MeGpppA_2'OMeG, at the 5' end. Two mRNA cap methyltransferase (MTase) activites involved in the formation of the cap, the (guanine-N7) and the (nucleoside-2'O)-MTases, reside in a single domain of non-structural protein NS5 (NS5MTase). We report on the biochemical characterization of the 2'OMTase activity of NS5MTase of dengue virus (NS5MTase_DV) using purified short capped RNA substrates (^7MeGpppAC_n or GpppAC_n). NS5MTase_DV methylates both types of substrate exclusively at the 2'O-position. The efficiency of 2'O-methylation does not depend on the methylation of the N7-position. Using ^7MeGpppAC_n and GpppAC_n substrates of increasing chain lengths, we found that both NS5MTase_DV 2'O-activity and substrate binding increased before reaching a plateau at n=5. Thus the cap and 6 nucleotides 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 ;micro;M, respectively). As other reported AdoMet-dependent RNA and DNA MTases, the 2'OMTase activity of NS5MTaseDV shows a low turnover of 3.25 x 10^-4 s^-1. Finally, we set up an inhibition assay and tested GTP- and AdoMet analogs as putative inhibitors of NS5MTase_DV. We confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (AdoHcy) (IC₅₀ 0.34 µM) and sinefungin (IC₅₀ 0.63 µM) demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.

Received 30 July 2009; accepted 22 September 2009.