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

Stewart, Meredith E.; Roy, Polly

Baculovirus expressing VP4 with site specific mutation as described above were generated as described [38] . Briefly, pAcYM1VP4 with the mutations and Bacmid: 1629 were transfected into a monolayer of Sf9 using Genejuice (Novagen) as per manufacturer protocols. After one round of virus amplification, plaque assay was performed and a number of virus isolates picked. These plaque purified viruses were propagated and screened for VP4 expression. Recombinant protein VP4 was purified from Sf9 cells infected with AcNPV expressing recombinant VP4 or VP4 mutants as described [33] . 1 Â 10 8 Sf9 were infected with recombinant baculovirus at MOI $ 5, cells were harvested at 68 h p.i and lysed in HNN buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 0.5% NP40); and lysate was centrifuged and pellet suspended in HN buffer (50 mM Hepes pH 7.5, 1 M NaCl). Supernatants were pooled and RNA precipitated with the addition of 0.1% polyethylenimine (PEI). VP4 was purified via size exclusion on a HiPrepS200 column. Fractions were collected and VP4 further purified using a heparin affinity column. VP4 was eluted from the column using a 250 mM to 2 M NaCl gradient in 25 mM Hepes pH 7.5.