FIG. 1.

(A) Schematic representation of the distribution of domains in the DENV NS5 protein. The FL NS5 protein has three major functional domains. The N-terminal MTase spans residues 1 to 296. The NLS has been divided into αNLS (spanning residues 320 to 368) and α/βNLS (residues 369 to 405). The arrow indicates the αNLS region thought to interact with the NS3 viral helicase. The six conserved amino acid sequence motifs within the RdRp domain (residues 273 to 900) are colored as in Fig. 2C, and “P” denotes the priming loop site. Also shown are the various truncated constructs of NS5 (see the text for details). (B) Schematic representation of the polymerase assay in the SPA bead format. The newly transcribed RNAs with the incorporation of radioactive GTP were captured on streptavidin-coupled beads and detected by counting as described in Materials and Methods. (C) Steady-state kinetics of RNA synthesis by DENV 3 RdRp, using a poly(C) primer and oligo(G) template. The reaction mixture contained 1 μM enzyme, 0.25 μg poly(C), 0.0125 μg oligo(G), 4 μM total GTP, and 0.5 μCi of [³H]GTP, as described in Materials and Methods. (D) Kinetic analysis of 3′dGTP inhibition. The IC₅₀ of 3′dGTP for DENV 3 RdRp was determined using at least two independent experiments. Assay conditions are described in Materials and Methods. (E) Limited proteolysis of the DENV 2 FL NS5 protein (left) and of the DENV 3 RdRp domain (right). Lanes 1 and 4, molecular mass markers; lanes 2 and 3, FL undigested NS5 and trypsin-digested NS5, respectively; lanes 5 and 6, undigested DENV 3 RdRp and trypsin-digested DENV 3 RdRp, respectively. The trypsin/enzyme ratio for FL NS5 was 1:100, and the reaction was carried out at room temperature for 2 h (lane 3). Similarly treated DENV 3 RdRp was completely digested (data not shown). Using a lower concentration of trypsin (1:1,000 ratio of trypsin to RdRp) for ∼30 min at room temperature yielded several smaller fragments (lane 6). The N termini of digested protein fragments were sequenced (indicated by arrows), and this information was used to derive some of the constructs shown in panel A.