Fig. 4.

Cap methylation analysis using tobacco acid pyrophosphatase. SeV rWT (“SeV mRNA”) and VSV rWT (“VSV mRNA”) were produced in vitro transcription by detergent-activated purified virions in the presence of [³H]AdoMet. Synthetic SeV NP mRNA controls were synthesized in vitro using T7 RNA polymerase and then used to make mRNA containing Cap 0 (m⁷GpppA...) labeled with [³H]AdoMet at the G-N-7 position, or mRNA containing Cap 1 (m⁷Gppp[m^2′-O]A...) labeled with [³H]AdoMet only at the 2′-O position using vaccinia virus enzymes as described in Materials and methods. For TAP analysis, all RNAs were normalized by [³H] counts and digested by TAP (“+TAP”) or mock-treated using the same reactions but without TAP (“-TAP”). mRNA was then separated from [³H]m⁷G using Sephadex G-50 mini-columns. [³H]Met incorporation into the G-N-7 or 2′-O cap positions was measured by scintillation counting of the entire flow through (for mRNA containing [³H-m^2′-O]A) and columns (for removed [³H]m⁷G). The data represent the mean ± standard deviation of two independent experiments.