In vitro oxidation assay was performed as described previously (18). Briefly, a reaction (100 μl total volume) containing 5 μg of substrates (purified RIG-I, KSHV helicase, or MCM7), 2 μg of enzymes (purified viperin or viperin-Del), 50 mM Hepes (pH 7.5), 150 mM KCl, 2 mM SAM, and 5 mM dithionite was incubated for 45 min at 37°C. Reaction was stopped by boil for 10 min. Reactions containing RIG-I, MCM7, and KSHV helicase were analyzed by mass spectrometry, and the ratio of oxidized peptides over total peptides was quantitatively determined.

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