B. G. Bradel-Tretheway and others Fig. 3. The human H5N1 3P complex is active at high temperatures and the H5N1 PB2 subunit confers this same phenotype on the WSN 3P complex. Equivalent amounts of polymerase complex (as determined by polymerase activity at 30 6C) were tested for transcriptional activity at different temperatures (30, 34, 37, 39, 42 6C). (a, b) Representative autoradiograms and quantitative data (top and bottom panel, respectively) for the ApG- (a) and the cap-RNA-primed (b) assays are shown. 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 data from at least four independent assays. Bars, SEM. An avian PB2 subunit renders a mammalian WSN complex transcriptionally more active In light of the putative avian origin of H5N1 viruses, such as A/Viet/1203/04, we wondered whether an avian PB2 (H3N2) protein might have similar properties to the human H5N1 PB2 protein. To test this hypothesis, we cloned and expressed the PB2 subunit from the A/chicken/ Nanchang/3-120/01 strain. We then purified 3P complexes (PA/PB1/PB2) from the WSN strain as well as complexes in which the PB2 subunit was substituted with an avian PB2 (W/W/N) or a mutated avian PB2 (W/W/NE627K). An E to 2928 K mutation in an avian PB2 has been shown to significantly enhance polymerase activity in mammalian cells, as measured in transcription–replication experiments (Labadie et al., 2007; Massin et al., 2001). The purified complexes were analysed by silver staining and by immunoblotting against PB2 (Fig. 4a top and bottom panel, respectively). All complexes were able to initiate transcription at 30 uC in a cap-independent and a cap- dependent fashion (Fig. 4b top and bottom panel, respectively). Complexes containing the avian PB2 subunit were found to have 20-fold greater activity than the WSN 3P complex in the ApG-primed and 10–20-fold greater activity in the globin-primed transcription assay. Interestingly, in both assays an E627K mutation in the avian PB2 resulted in a slight increase in activity. An avian PB2 confers broad temperature tolerance on the WSN 3P complex The purified complexes (W/W/W, W/W/N) were evaluated for their activity over a range of temperatures by using an equivalent amount of functionally active complex as described above. In the ApG- and cap-primed transcription assay (Fig. 5a and c) the W/W/N complex had optimal activity at 30–39 uC (peaking at 37 uC and modestly declining at 42 uC). We then quantified the transcriptional activities of all three complexes (W/W/W, W/W/N, W/W/ NE627K) in the ApG- and cap-primed transcription assay (Fig. 5b and d, respectively) and found, that an E627K mutation slightly shifted the temperature optimum from 37 uC to 39 uC. Collectively, these results suggest that an avian PB2 subunit behaves similarly to the VN1203 (H5N1) PB2 subunit, and that it too enhances the activity of the WSN 3P complex at elevated temperatures. An avian PB2 confers enhanced functional stability on the WSN 3P complex To investigate the mechanistic basis for the increased thermal tolerance of complexes containing the avian PB2 subunit, we conducted a comparative analysis of the thermostability of the W/W/W and the W/W/N complexes. For this experiment, an equal amount of 3P complexes (as determined by functional activity at 30 uC) was preincu- bated at 30 uC. This occurred in the absence of any vRNA or nucleosides over different time periods (0–120 min). The transcriptional activity of each complex was then measured (30 uC, 1 h). It has been reported that the WSN 3P complex is heat labile in the absence of vRNA (Brownlee & Sharps, 2002), which was confirmed by our analysis. For the W/W/W complex we observed an exponential decay in transcriptional activity when pre-incubated at 30 uC (Fig. 6a, b); the half-life of the complex was estimated at 5–12 min at this temperature (Fig. 6b). However, the W/W/N complex was very stable over the entire time- period – with a loss of activity of only about 25 % over a Journal of General Virology 89