FIG 8.

Sensitivity of wt SFV nsP1 and SFV nsP1 K231R+K300E to inhibition by SAH and dependence on SAM. (A) wt SFV nsP1 and the double mutant were incubated with [α-³²P]GTP and 10 μM SAM in the presence of 0 to 1 mM SAH at 30°C for 30 min, and then the reaction was stopped with 10% SDS. (B) wt SFV nsP1 and double mutant were incubated with [α-³²P]GTP and 0 to 10 μM SAM at 30°C for 30 min, and then the reaction was stopped with 10% SDS. In both panels A and B the α³²P-m⁷GMP-nsP1 covalent intermediate was visualized after overnight exposure of the phosphorimager screen. Coomassie blue staining was used to demonstrate loading of equal protein quantities. The relative inhibition by SAH (indicated as the percentage of untreated control below the lanes in panel A) of both wt SFV nsP1 and SFV nsP1 double mutant were calculated using QuantityOne software by dividing the volume of the bands of interest by the untreated control. The relative activities at various concentrations of SAM expressed as percentages of the activity at 10 μM SAM are indicated below each lane in panel B.