B. G. Bradel-Tretheway and others Fig. 6. An avian PB2 subunit confers enhanced thermostability on the WSN 3P complex. Equivalent amounts of polymerase complex (as determined by polymerase activity at 30 6C) were pre- incubated at 30 6C in the absence of vRNA for different time periods. The complexes were then tested in an ApG-primed transcription assay (30 6C, 1 h). (a) A representative autoradio- gram and (b) quantitative data from replicate experiments are shown. The data were normalized to the amount of fully extended product where no pre-incubation was performed (as quantified by Phosphorimager analysis). Mean transcriptional activities from at least four independent assays are shown. Bars, SEM. A nonlinear curve for a single-phase exponential decay was fitted to the data using GraphPad Prism. The r2 value (goodness of fit) for the W/W/ W curve was 0.81, and the half-life of the W/W/W complex was estimated at 7.3 min (with 95 % confidence intervals ranging from 5.3 to 11.8 min). The half-life of the W/W/N complex could not be determined, as it exceeded the 2 h of our analysis. (c) The purified WSN 3P complex was pre-incubated at the indicated tempera- tures in the presence (left panel) or absence of both 59- and 39- vRNA (right panel). The proteins were pulled-down with NiNTA Magnetic Agarose Beads and detected by immunoblotting with antibodies directed against PA (anti-His), PB1 and PB2. DISCUSSION Some H5N1 avian influenza viruses (Claas et al., 1998; Li et al., 2004) exhibit high pathogenicity in humans with pandemic potential (Gambotto et al., 2008). This under- scores the need to better understand the mechanisms that determine the ability of emerging influenza viruses to cross 2930 the species barrier into humans. This process is regulated in part by the viral haemagglutinin (HA) and host sialic acid receptors (Suzuki et al., 2000). The viral polymerase complex is also recognized to play an important role in IAV host-cell specificity and pathogenicity (Gabriel et al., 2005, 2007; Labadie et al., 2007; Massin et al., 2001; Salomon et al., 2006), but the underlying basis for this remains poorly understood. In this report, we show that the IAV 3P complex can be successfully purified from human lung epithelial cells using an adenoviral system. We analysed the functional activity of IAV polymerase complexes from the WSN (H1N1) and VN1203 (H5N1) strains, as well as chimeric complexes in which the PB2 subunits were exchanged. Our data show that the H5N1 polymerase complex has more robust thermotolerance than its WSN counterpart, which is active over a narrow temperature range (30–34 uC) with a more than 50 % reduced activity at 37 uC. This effect can be attributed to the PB2 subunit, since a simple exchange of this protein was associated with substantial changes in the temperature sensitivity of the polymerase activity. This effect does not depend on the presence of a glutamic acid residue at position 627, since the PB2 subunits from both the VN1203 and the WSN strain harbour a lysine at position 627 (K627). This suggests that additional determi- nants within PB2 contribute to virus host-range and replication efficiency as well (Gabriel et al., 2005; Naffakh et al., 2000; Shinya et al., 2004). We also generated complexes in which we substituted the PB2 subunit in the WSN 3P complex with an avian (H3N2) PB2 counterpart. Functional analysis of these complexes revealed that, like the H5N1 PB2 subunit, the avian PB2 subunit also conferred improved thermotoler- ance on the WSN 3P complex. Associated host-factors could possibly contribute to the functional properties of the purified 3P complex, although future experiments will be needed to address this question. Our analyses also showed that the E627 residue in the avian PB2 was not required for the enhanced functional activity of the chimeric W/W/N. The E627K mutation in the avian PB2 led to only a slight increase in thermotolerance. Studies that have described the role of PB2 amino acid residue 627 as a determinant of cold sensitivity of avian influenza viruses were performed using transcription– replication experiments (Labadie et al., 2007; Massin et al., 2001) or by studying the replication of infectious, recombinant viruses (Hatta et al., 2007; Salomon et al., 2006). These assays effectively examine both RNA tran- scription and genome replication, since a defect in either process would lead to a reduction in measured overall replication. In contrast, our experiments examined only viral transcription. Since viral transcription and replication occur via distinct mechanisms, our findings suggest that PB2 residues other than amino acid 627 may regulate temperature sensitivity of viral transcription – and that mutations at these residues may contribute to virus-host Journal of General Virology 89