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A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses.

Lim, Siew Pheng, Wen, Daying, Yap, Thai Leong, Yan, Chung Ka, Lescar, Julien and Vasudevan, Subhash (2008) A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses. Antiviral research, 80 (3). pp. 360-369. ISSN 1872-9096


Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity at its N-terminal amino acid sequence and is responsible for formation of a type 1 cap structure, m(7)GpppAm(2'-O) in the viral genomic RNA. Optimal in vitro conditions for DENV2 2'-O-MTase activity were characterized using purified recombinant protein and a short biotinylated GTP-capped RNA template. Steady-state kinetics parameters derived from initial velocities were used to establish a robust scintillation proximity assay for compound testing. Pre-incubation studies showed that MTase-AdoMet and MTase-RNA complexes were equally catalytically competent and the enzyme supports a random bi bi kinetic mechanism. The assay was validated with competitive inhibitory agents, S-adenosyl-homocysteine and two homologues, sinefungin and dehydrosinefungin. A GTP-binding pocket present at the N-terminal of DENV2 MTase was previously postulated to be the cap-binding site. Interestingly, inhibition of the enzyme by GTP was two-fold lower than with RNA cap analogues, G[5']ppp[5']A and m(7)G[5']ppp[5']A and about three-fold poorer than a two-way methylated analogue, m(7)G[5']ppp[5']m(7)G. This assay allows rapid and highly sensitive detection of 2'-O-MTase activity and can be readily adapted for high-throughput screening for inhibitory compounds. It is suitable for determination of enzymatic activities of a wide variety of RNA capping MTases.

Item Type: Article
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Additional Information: author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: Dengue; Methyltransferase; Assay; Kinetics; Compound; Inhibition
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Date Deposited: 14 Dec 2009 14:02
Last Modified: 31 Jan 2013 01:21


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