FIG 5.
Effects of nsP2 mutations on the RNA helicase, ATPase, and protease activities of the purified recombinant proteins. (A) RNA helicase activities of nsP2 and mutants were analyzed using the Alexa488-dsRNA28/16 substrate with a 12-base 5′ overhang. The band intensities were quantified by ImageJ. Alexa Fluor 488 is indicated by a black star. (B) ATP hydrolysis was carried out at various ATP concentrations. (C) Protease activities were determined by use of a FRET-based octapeptide substrate [DABCYL-RAGG↓YIFS-(Glu-EDANS)-NH2]. All enzymatic assays were performed in triplicate, and the data are means and SD. Significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s posttest comparing mutants to nsP2 (vehicle control). ***, P < 0.001; ns, not significant.