Fig. 4.
An avian PB2 subunit increases the activity of the WSN 3P complex at 30 °C. (a) Purified 3P complexes from the WSN (W/W/W) virus were prepared, along with complexes in which the PB2 subunit was omitted (W/W) or substituted by the PB2 from the avian Nanchang virus (W/W/N) or a mutated derivative (W/W/NE627K). The purified complexes were detected by silver staining and immunoblotting against PB2 (top and bottom panel, respectively). PB2-normalized complexes were used in functional assays (30 °C). (b) The complexes were analysed for cap-independent (ApG) or cap-dependent transcription (top and bottom panel, respectively). The transcription products in the ApG primed assay (14 nt long transcript) and in the globin-primed assay (triplet band; capped primer +14 nt) were detected by autoradiography. (c, d) Quantitative data for the ApG- and the cap-RNA-primed assays are shown. The data were normalized in terms of fold activity over the activity of the W/W/W complex. The results shown represent mean transcriptional activity from at least three independent assays. Bars, sem.