FIG 1.

ADP-ribosyl-binding and hydrolase activities of SINV nsP3^MD mutants. (A) Representative image of results from the PARP10 catalytic domain (PARP10^CD) demodification assay. PARP10^CD was incubated with ³²P-NAD⁺ to generate ³²P-MARylated PARP10^CD, which was incubated with buffer alone, nsP3 MDs from WT and mutants for 1 h at 37°C, followed by analysis by SDS-PAGE and autoradiography. Changes in the intensity of ³²P-MARylated PARP10^CD in samples containing nsP3^MD from WT and mutants were quantified. (B) Quantitative representation of MAR hydrolase activity of nsP3 MD mutants relative to WT. Assays were performed in triplicate, buffer control was subtracted, and values were normalized to the activity levels of nsP3 MD WT. The data are presented as the percent WT activity values obtained from three independent experiments. Significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test. ****, P < 0.0001 (WT versus N24A, G32E, TM [G32E/I113R/Y114N], and Y114A). (C) Quantification of ADPr-binding in K_D (μM) from three runs of microscale thermophoresis (MST). Defined length PAR labeled on the 1″ terminus with Cy5 (10 nM) was incubated with 2-fold serial dilutions (diluted down from 0.5 to 1 mM stock concentration to 15 to 30 nM) of SINV WT and mutant MDs. MST was measured using a Monolith NT.115 (NanoTemper) at 80% excitation power and 20% MST power. The data are shown as the mean normalized fluorescence ± the SD.