Fig. 5.

T98M mutation causes nsp15 protein instability. (A) BMDMs infected with WT or N15m1 virus at an MOI of 0.1 were lysed at 16 hpi and viral N protein, nsp15, and β-actin were detected by Western blotting. (B) WT and the T98M mutant of nsp15 were expressed and purified from E. coli. Coomassie blue staining shows the purified SUMO-tagged WT and T98M nsp15, which were detected by anti-nsp15 antibody using Western blotting (Lower). (C) DSF thermal shift analysis of nsp15 WT (black) and nsp15-T98M mutant (red) proteins. (D) A radiolabeled RNA molecule R16.4 was treated over time with WT nsp15 or T98M in the presence of 5 mM Mn²⁺. At the indicated time points, an aliquot of the reaction was analyzed on a denaturing 20% polyacrylamide gel. The sequence of RNA R16.4 is shown above the gel image. The only uridylate, at position 13, is underlined. Data are representative of two to three independent experiments.