Fig 6. Stabilization and inhibition of ANDV endonuclease by a known endonuclease inhibitor.
A, Thermal stability of ANDV L1–200 proteins in the presence of 16 mM MnCl2 and 100 μM 2,4-dioxo-4-phenylbutanoic acid (DPBA) measured in thermofluor assay (Tm, melting temperature). B, RNA nuclease assay with ANDV L1–200 N167A in the presence of 2 mM MnCl2 and increasing concentrations of DPBA. The inhibitor was incubated with the enzyme for 15 min prior to the addition of the 27mer ssRNA substrate. The assay was performed for 1 h at 37°C. Substrate and reaction products were separated on a denaturing polyacrylamide gel. C, Signals for residual uncleaved substrate as shown in panel A were quantified using a phosphorimager.