FIG 3.

Inhibitory effects of selected compounds on the enzymatic activity of purified wt SFV or CHIKV nsP1 in a biochemical assay measuring the formation of the covalent [³²P]m⁷GMP-nsP1 reaction intermediate. (A) wt SFV nsP1 was incubated with [α-³²P]GTP and 100 μM SAM and was treated with increasing doses (50 μM to 1 mM) of inhibitors. SFV nsP1 D64A was used as a negative control. VC, solvent control. (B) wt SFV nsP1 was incubated with [α-³²P]GTP with or without 50 μM sinefungin (SIN) in the presence or absence of 100 μM SAM. SFV nsP1 D64A was used as a negative control. (C) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM and was treated with increasing doses (0.5 to 32 μM) of inhibitors. (D) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM and was treated with increasing doses (125 μM to 1 mM) of sinefungin. (E) wt CHIKV nsP1 was incubated with [α-³²P]GTP and 10 μM SAM in the presence (left) or absence (right) of DTT and increasing concentrations (12.5 to 500 μM) of FHNA. In all cases, the covalent [α-³²P]m⁷GMP-nsP1 intermediate was visualized after overnight exposure of the PhosphorImager screen. Coomassie blue staining with GelCode blue reagent was used to demonstrate the loading of equal protein quantities.