Detection of hepatitis C virus helicase activity using the scintillation proximity assay system.
The C-terminal two-thirds of the nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in the viral replication and is expected to be one of the target molecules of anti-HCV drugs. The conventional method for the measurement of RNA helicase activity includes the step of gel electrophoresis which makes the screening of multiple samples inconvenient. In this study, to establish a high-throughput screening system for HCV helicase inhibitors, we applied the scintillation proximity assay (SPA) system to the detection of this enzymatic activity. We could detect the helicase activity using the NS3 protein purified by an immunoaffinity column. The activity was dependent on the concentration of the enzyme and the reaction time. The RNA helicase activity measured by the SPA system was in a good correlation with that obtained by the conventional method. Furthermore, the SPA system showed better reproducibility and less deviation of the data than the conventional method, which makes the former suitable for quantitative analysis. Since any separation step is not required and microtiter plates can be used in this method, it has the advantage of dealing with multiple samples.
Kyono
K
K
Lead Generation Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka, Japan.
Miyashiro
M
M
Taguchi
I
I
D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012588:Scintillation Counting; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011933:Reagent Kits, Diagnostic; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases