FIG 2.

Protein expression and in vitro enzymatic activities of DENV3 NS5 and its mutants. (A) SDS-12% PAGE of purified WT and mutant NS5 proteins purified as described previously (19); melting temperature values measured by Thermofluor assay are indicated in parentheses. Numbers at left are molecular masses in kilodaltons. (B) A de novo initiation/elongation assay was performed using viral untranslated region (UTR) sequence as the template as described previously (19, 20). (C) Elongation assays of DENV3 WT and mutant NS5 proteins were performed with a heteropolymeric RNA template annealed with four primers as described previously (10, 13). (D) N7 MTase activities of the WT and NS5 mutant were measured with the in vitro-transcribed first 110 nucleotides (nt) of the DENV genome with unmethylated cap G (12, 21). (E) 2′-O-MTase activities of DENV3 WT and mutant NS5 proteins were measured with a 7-mer single-stranded RNA with cap 0 structure (12, 21). Two independent experiments were performed for each assay in triplicate (RdRp assays) or duplicate (MTase assays). Percent activities relative to DENV3 WT NS5 are shown above each bar.