Paramyxovirus Polymerase Assays

Unless otherwise specified, all NiV and RSV polymerase reactions consisted of 0.2 μM oligonucleotide template derived the NiV leader promoter, 0.2 μM recombinant L-P complex, with a buffer containing 20 mM Tris pH 7.5, 10 mM KCl, 2 mM DTT, 0.5% triton, 10% DMSO, 6mM MgCl₂. Recombinant RSV L-P was produced through the co-expression of RSV L and P proteins in a baculovirus expression system, according to previously described procedures [23 (link)]. In the primer-dependent reaction, this was then combined with 200 μM primer. Reactions were initiated through addition of specific nucleoside TP for the template sequence to final volume of 10 μL and incubated at 30°C for 30 minutes. The radioisotope tracer in these reactions was α³³P-GTP. Reactions were stopped with the addition of an equal volume of gel loading buffer (Ambion, Austin, TX) denatured at 95°C for 5 minutes, and run on a 22.5% polyacrylamide urea sequencing gel for 2 hours at 80W. The migration products were exposed to a phosphor-screen, scanned on Typhoon phosphorimager (GE Healthcare, Chicago, IL) and quantified using ImageQuant (GE Healthcare).