Extended Data Fig. 2: RdRp activity assay.

a, SDS–PAGE of HMPV wild-type L:P, L(D745A):P purified for RdRp activity assays. Proteins were purified by metal affinity, TEV cleavage of the His-tag followed by reverse His-tag affinity purification and size-exclusion chromatography. b, Analysis of the 3′ extension activity of HMPV polymerase using the le25 RNA template. Reactions were performed with rNTPs (0.5 mM each of rUTP, rGTP and rCTP), 20 μM rATP and 20 nM [α-³²P]rATP. When a 3′-modified le25 (le25[SpC3], three-carbon spacer group linked to the 3′ extremity) was used as a template, synthesis of products longer than 25 nt was greatly reduced compared to le25. When only [α-³²P]rATP and no other rNTP was supplied, only a product with a length longer than 25 nt was observed. This result shows that the L:P complex was capable of modifying the 3′ terminus of the template, in addition to engaging in de novo initiation at the promoter. The radiolabelled RNA products were visualized by phosphor imaging. Data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.