Role of PB2 in IAV temperature sensitivity Fig. 2. The H5N1 3P complex is more active than the WSN 3P complex in both cap- independent and cap-dependent transcription. (a) Purified 3P complexes from the VN1203 (H/H/H) and WSN (W/W/W) viruses, along with chimeric (H/H/W and W/W/H) and dimeric complexes (H/H; negative control), 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 data from at least three independent assays. Bars, SEM. complexes containing avian subunits have elevated in vitro activity compared with those containing human IAV proteins (Taubenberger et al., 2005). The biological significance and mechanistic underpinnings of this obser- vation will require further investigation. The VN1203 polymerase is active at elevated temperatures, and the PB2 subunit is sufficient to confer broad temperature tolerance on the WSN polymerase The purified polymerase complexes were subjected to the ApG- (Fig. 3a) or globin-primed (Fig. 3b) transcription assay. Reactions were run at 30 uC and at a range of temperatures selected on the basis of their physiological relevance to influenza virus replication (34, 37, 39 and 42 uC). In these experiments, the amount of input 3P complex was standardized by using an equivalent amount of functionally active complex, as determined on the basis of transcriptional activity at 30 uC. The WSN complex (W/W/W) extended the ApG primer best at temperatures ranging from 30 to 34 uC; activity was reduced by more than twofold at 37 uC and more than tenfold at elevated temperatures (39–42 uC) (Fig. 3a). In contrast, the VN1203 complex (H/H/H) extended the primer best at 34–39 uC, and retained 25 % of its activity at elevated temperature (42 uC). Chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) exhibited a temperature sensitivity profile similar to that of the WT complexes from which the PB2 subunit was derived – i.e. the H/H/W http://vir.sgmjournals.org complex was minimally active at elevated temperatures, while the W/W/H complex retained a high level of functionality at 39–42 uC (Fig. 3a). In contrast, the presence of excess PA/PB1 heterodimers in the WSN complexes (W/W/W) had no effect on the thermotolerance of the polymerase (Supplementary Fig. S1). Results from the cap-primed transcription assay (Fig. 3b) were essentially equivalent to those obtained from the ApG-primed assay. The W/W/W complex extended the capped mRNA best at 30 uC, and its activity was reduced to 50 % at 34–37 uC and to less than 20 % at 39–42 uC. In contrast, the H/H/H complex extended the capped primer best at 30–39 uC, and retained significant activity at 42 uC. In this assay, the temperature sensitivity profile of the activity of the chimeric complexes was again determined by the PB2 subunit. Overall, results obtained with the cap-independent and cap-dependent transcription assays were very similar. One minor difference was that, in the ApG-primed transcription assay, we observed a somewhat more pronounced difference in temperature sensitivity between complexes harbouring the WSN PB2 subunit (W/W/W, H/H/W) and complexes containing the VN1203 PB2 protein (H/H/H, W/W/H). Collectively, these results showed that the VN1203 complex is active at a broad range of temperatures, that this property is dependent on the PB2 subunit, and that this phenotype can be transferred to the WSN 3P complex simply by exchanging the PB2 subunit. 2927