Recombinant RSV Polymerase Assay

The recombinant RSV polymerase complex was produced by co-expressing the L and P proteins of RSV in insect cells as previously described [25 (link)]. Unless otherwise specified, RNA polymerase reaction samples consisted of 0.2 μM of an oligonucleotide template sequence derived from the RSV leader promoter (5'-UUUGUUCGCGU-3') and 0.2 μM recombinant RSVL-P polymerase together with 200 μM 5'-pACGC primer, mixed in a buffer containing 20 mM Tris pH 7.5, 10 mM KCl, 2 mM dithiothreitol, 0.5% triton, 10% DMSO, 0.2 U/μL RNasin (Ambion), and 6 mM MgCl₂. Reactions were started at 30°C by adding specific NTPs in a final volume of 10 μL. The radioisotope tracer used for this assay was α³³P-GTP. Reactions were stopped after 30 minutes by adding an equal volume of gel loading buffer (Ambion). Samples were denatured at 95°C for 5 minutes, and run for 1.5 hours at 80 W in a 22.5% polyacrylamide urea sequencing gel. After the gel was dried, the product of migration was exposed to a phosphor-screen and scanned as previously described.