In Vitro Biochemical Assays to Study Respiratory Syncytial Virus Polymerase Activities

Here, we detail a biochemical assay that assesses respiratory syncytial virus (RSV) RNA-dependent RNA polymerase activities at the viral promoters in vitro. The assay consists of a purified recombinant RSV polymerase complex comprising the L protein and its cofactor P, combined with a short RNA template and radiolabeled NTPs. RNA synthesis by the L-P complex yields radiolabeled RNA products that can be separated at the nucleotide level on denaturing polyacrylamide gels and detected by autoradiography or phosphor image analysis. Each component of the assay can be readily manipulated. As such, this simple assay has aided the understanding of early polymerase transcription and genome synthesis initiation events, revealed previously unknown polymerase functions, and determined how various RSV polymerase inhibitors function.