Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

Methyl transfer from Ado[methyl-3 H]Met to T7 derived BTV ssRNAs were used to determine the ability of the methylase activity of mutant VP4. A standard reaction mixture contained 50 mM TrisHCl (pH 7.5), 9 mM MgCl 2 , 2 mM Ado[methyl-3 H]Met (87 Ci/mmol), 6 mM DTT, 1 lg of uncapped ssRNAs or 0.4 mM Cap analogue (GpppA, m7 GpppG; NEB) and 2 lg purified protein. After incubation at 37°C for 2 h, the reaction mixtures were treated with proteinase K. RNA/cap analogues were purified by phenol:chloroform extraction and precipitated with cold ethanol. Cap analogues were precipitated in 9 vol ice-cold ethanol. The transfer of the 3 H-methyl was determined using PerkinElmer 1450 microbeta. As a negative control baculovirus infected cytosolic lysate was used, which was extracted from infected Sf9 cells using HNN buffer as described for VP4 recombinant proteins, treated with PEI and centrifuged to remove contaminating RNA and baculovirus particles. Reactions were performed in triplicate and data were analysed for significance using student t-test.