Fig. 1: Characterization of purified L proteins.
a Chemical structure of MRK-1. b Schematic diagram illustrating the domain organizations of full-length RSV and HMPV L proteins and truncated RSV L protein (Ltrunc). Flexible domains not captured in pneumovirus L structures are colored in gray. c SDS-PAGE of purified L-P and Ltrunc-P proteins migrated alongside a BenchMark protein ladder (Invitrogen). d Titration of unlabeled MRK-1 against radiolabeled MRK-1 to determine the affinity of binding to wild-type (WT) RSV L-P. The data show the mean and data points for three technical replicates, representative of one of three independent experiments (see also Supplementary Fig. 1a, c). Dissociation constant (KD) is denoted under the curve. e Analysis of competition between unlabeled NNIs and radiolabeled MRK-1 for binding to WT RSV L-P. Data show the mean and data points for three technical replicates, representative of one of three biological replicate experiments (see also Supplementary Fig. 1b, d). f Schematic diagram illustrating the design of the RNA synthesis assay. g RNA synthesis activities of RSV L-P and Ltrunc-P from position 3C of the RSV trailer (tr) promoter. A phosphorimage representing one of three independent experiments is shown. Lane 1 shows a ladder representing products generated from position 3C of a trailer 1-25 promoter. Note that the ladder RNAs have a 5′ monophosphate and products ≤6 nt in length migrate differently than RNA with a 5′ triphosphate. Lane 2 shows a marker for the pppGpA dinucleotide.
