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 hepa titis C virus (HCV) possesses RNA helicase activity. This enzyme is co nsidered to be involved in the viral replication and is expected to be one of the target molecules of anti-HCV drugs. The conventional metho d for the measurement of RNA helicase activity includes the step of ge l electrophoresis which makes the screening of multiple samples inconv enient. In this study, to establish a high-throughput screening system for HCV helicase inhibitors, we applied the scintillation proximity a ssay (SPA) system to the detection of this enzymatic activity. We coul d detect the helicase activity using the NS3 protein purified by an im munoaffinity column. The activity was dependent on the concentration o f 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 reprod ucibility 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 t his method, it has the advantage of dealing with multiple samples. (C) 1998 Academic Press.