Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors

► Wheat-germ cell-free protein system simplified the production of DENV NS3 and NS5. ► A robust assay for NS3-NS5 interaction was established by AlphaScreen technology. ► Protease and helicase domains of NS3 were required for the binding to NS5. ► Both domains of NS3 exhibited competitive actions to the NS3-NS5 interaction.