Role of PB2 in IAV temperature sensitivity Fig. 4. An avian PB2 subunit increases the activity of the WSN 3P complex at 30 6C. (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 deriv- ative (W/W/NE627K). The purified complexes were detected by silver staining and immuno- blotting against PB2 (top and bottom panel, respectively). PB2-normalized complexes were used in functional assays (30 6C). (b) The complexes were analysed for cap-independent (ApG) or cap-dependent transcription (top and bottom panel, respectively). The transcrip- tion 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. 2 h period. These results demonstrate that the avian PB2 subunit confers greater stability on the WSN 3P complex and extends the functional half-life of the complex. While the functional activity of the W/W/W complex rapidly declined during incubation at 30 uC, this could not be attributed to simple physical dissociation of the complex, since it remained intact even in the absence of vRNA (Fig. 6c). http://vir.sgmjournals.org Fig. 5. An avian PB2 subunit confers broad temperature tolerance on the WSN 3P com- plex independently of amino acid residue 627. Purified 3P complexes were prepared from the WSN virus (W/W/W), along with complexes in which the PB2 subunit was substituted by an avian PB2 (W/W/N) or a mutated derivative (W/W/NE627K); an extract prepared from LacZ-expressing cells (LacZ) was used as a negative control. Equivalent amounts of poly- merase complex (as determined by polymerase activity at 30 6C) were tested for transcrip- tional activity at different temperatures (30, 34, 37, 39, 42 6C). Representative autoradio- grams (a, c) and quantitative data (b, d) are shown for the ApG- (a, b) and the cap-RNA- primed (c, d) assays. The data were normalized in terms of percentage of the amount of fully extended product at 30 6C (as quantified by Phosphorimager analysis); the results shown represent mean transcriptional activity from at least four independent assays. Bars, SEM. 2929