A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses.
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.
Lim
Siew Pheng
SP
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670, Singapore. siew pheng.lim@novartis.com
Wen
Daying
D
Yap
Thai Leong
TL
Yan
Chung Ka
CK
Lescar
Julien
J
Vasudevan
Subhash G
SG
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D003716:Dengue Virus / Q000737:chemistry* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D012588:Scintillation Counting / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D014764:Viral Proteins; D006160:Guanosine Triphosphate; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; D000241:Adenosine; C006235:sinefungin