Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening

Abstract

Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1-4 FL NS5. Heavy metal ions such as Zn(++) and Co(++) as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.

Keywords: Activity; Assay; Compounds; De novo initiation; Dengue virus NS5 protein; Elongation; Flavivirus; Inhibition; Kinetics; RNA dependent RNA polymerase.