MERS-CoV and SARS-CoV nsp14 Methyltransferase Assay

Methyltransferase assays were performed in 40 mM Tris-HCl (pH 8.0), 5 mM DTT, 2 μM ^7MeGpppA or GpppA RNA cap analogue (New England Biolabs), 10 μM adenosyl-methionine (AdoMet; Thermo Fisher), 0.03 μCi/μl [³H]AdoMet (PerkinElmer) (25 (link)). For each reaction, MERS-CoV or SARS-CoV nsp14 was added to a final concentration of 500 or 250 nM, respectively. Reaction mixtures were incubated at 30°C for up to 120 min, and reactions were stopped by the addition of a 10-fold volume of 100 μM ice-cold adenosyl-homocysteine (AdoHcy; Thermo Fisher). Then, samples were spotted on a DEAE filter mat (PerkinElmer) prewet with Tris-HCl (pH 8.0) buffer. Filter mats were washed twice with 10 mM ammonium formate (Sigma-Aldrich) (pH 8.0), twice with MilliQ water, and once with absolute ethanol (Sigma-Aldrich). After air drying for 10 min, filter mats were cut, and relevant pieces were transferred to individual tubes. Betaplate scintillation fluid (PerkinElmer) was added, and the amount of ³H label bound was measured in counts per minute using a Wallac scintillation counter. For relative quantification, incorporation measurements for mutant proteins were normalized to values obtained with the wt control nsp14. Samples were measured in duplicate in each experiment.