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

Abstract

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 7GpppAm 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 7G[5′]ppp[5′]A and about three-fold poorer than a two-way methylated analogue, m 7G[5′]ppp[5′]m 7G. 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.