B. G. Bradel-Tretheway and others (Hatta et al., 2007; Massin et al., 2001; Murakami et al., 1988; Torreira et al., 2007): the presence of lysine at position 627 (K627), instead of the glutamic acid (E627) that is found in avian viruses, is crucial for high virulence in mice (Hatta et al., 2001). This amino acid does not determine the cell tropism of the virus (Shinya et al., 2004), but instead enhances viral growth in mice and probably in humans (Hatta et al., 2007; Salomon et al., 2006). The mechanism underlying this observation still needs to be elucidated. Most studies on temperature sensitivity of IAVs have been performed at the level of viral transcription-replication. We were interested in how different temperatures directly affect viral RNA polymerase activity at the transcriptional level. To do this, we elected to use two well-characterized IAVs: (i) the mouse-adapted A/WSN/33 H1N1 virus (‘WSN’) and (ii) the A/Viet/1203/04 H5N1 virus (‘VN1203’). WSN has been described to replicate with about tenfold lower efficiency at 39.5 uC compared with 34 uC, as measured by plaque assay (Kawaguchi et al., 2005). In contrast, the VN1203 virus has been shown to replicate efficiently at both low (34 uC) and high temperatures (41 uC) (Hatta et al., 2007). We purified 3P complexes (PA/PB1/PB2) from the VN1203 (H/H/H) and WSN (W/W/W) strains and compared the effect of temperature on cap-independent and cap-dependent transcription. In addition, we exchanged the PB2 subunits between these two complexes (H/H/W, W/W/H) or substituted the PB2 subunit with an avian subunit from the A/chicken/Nanchang/3-120/01 strain (H3N2) (W/W/N). We developed an adenoviral expression system which is attractive, because recombinant adenoviruses can enter a broad range of cells, including IAV-susceptible human lung epithelial cells (A549). As a result, it is possible to prepare authentic IAV polymerase complexes that contain associated host cell factors that participate in complex formation and/or polymerase function. Using this approach, we show here that PB2 directly affects the temperature optimum for IAV tran- scription, and that incorporation of an H5N1 or avian PB2 subunit into the IAV 3P complex results in greatly increased polymerase activity at elevated temperatures characteristic of birds. We also show that this effect of the H5N1 and avian PB2 subunits does not depend on the presence of a glutamic acid residue at position 627 of PB2. Finally, our results suggest that PB2 may exert its effect on the temperature optimum of the IAV RNA polymerase by influencing the functional stability of this trimeric protein complex. METHODS Influenza virus strains, vectors and cells. Each polymerase subunit of the WSN (H1N1) strain (A/WSN/33) was subcloned from pcAGGS-PA/PB1/PB2WSN (gift of Dr Y. Kawaoka, University of Wisconsin-Madison, WI, USA). The PB2 gene was amplified by reverse-transcription PCR from the Nanchang (H3N2) strain (A/ 2924 chicken/Nanchang/3-120/01), obtained from Dr R. Webster, St Jude Children’s Research Hospital, Memphis, TN, USA (Liu et al., 2003). The E627K mutation was introduced by site-directed mutagenesis (Quick-Change mutagenesis kit, Stratagene). The polymerase genes of the VN1203 (H5N1) strain (A/Vietnam/1203/04) were assembled from synthetic oligonucleotides by GENEART (GenBank accession nos EF467808, AY818132.1, AY651719.1). PA from the WSN and the VN1203 strain were tagged at the C terminus with a tandem-affinity purification (TAP)-tag. The TAP-tag was constructed by overlapping PCR-mutagenesis, resulting in a C- terminal TAP where the thrombin cleavage site was followed by a 66His tag, a tobacco etch virus (TEV) cleavage site and an IgG- binding domain (protein A domain). Briefly, the ZZ domain of BG1766 (Malkowski et al., 2007) was amplified by overlapping PCR introducing a TEV cleavage site. The amplified PCR product was ligated at the 59-terminal EcoRV site to a dsDNA fragment (TCTAGACTGGTGCCGCGCGGCAGCCATCATCATCACCATCAT- GATATC) containing a thrombin site and a 66His tag. WSN or VN1203 PA were fused at their C terminus to the TAP-tag. Human embryonic kidney (HEK293A or QBI-HEK 293CymR) or human lung endothelial cells (A549) were grown at 37 uC and maintained in DMEM supplemented with 10 % fetal bovine serum, 50 IU penicillin ml21, 50 mg streptomycin ml21 and 2 mM glutamine. Adenovirus production. Recombinant adenoviruses expressing either LacZ or PB2 from the Nanchang strain (N or NE627K) or PA- TAP, PB1 or PB2 from the WSN or the VN1203 strain were created from pShCMV by homologous recombination into AdEasy and propagation in HEK293A cells (AdEasy system, Stratagene). Only the adenovirus expressing PB2 from the VN1203 strain was constructed differently, by cloning PB2 into the pAdenoVator-CMV(CuO) plasmid followed by recombination into AdEasy and propagation in QBI-HEK 293CymR cells (Qbiogene). The viruses were purified by CsCl density-gradient centrifugation and dialysis. Viral titres were determined by quantitative real-time PCR (Heim et al., 2003). Polymerase purification. A549 cells (66106 in a T-225 flask) were transduced the following day with each of the three adenoviruses (rAd5-PB1, -PB2, -PA-TAP) at an m.o.i. of 1000 (80 % transduction efficiency); for adenoviruses expressing the codon-optimized genes PA, PB1 and PB2 from the VN1203 (H5N1) strain an m.o.i. of about 100 was used. Nuclear and cytoplasmic extracts were prepared 48 h post-infection (Nuclear Extract kit, Active Motif) and purified using the TAP method (described below). Lysates from whole cells were prepared 48 h post-infection in 3 ml ice-cold 16 RIPA buffer (Upstate) supplemented with 1 mM NaF, 1 mM PMSF, 1 mM NaVO3, 1 ml Protease Inhibitor Cocktail (P8340, Sigma). Poly-Prep Chromatography Columns (0.864 cm, Bio-Rad) were loaded with 300 ml IgG Sepharose 6 fast flow bead suspension (Amersham) and washed with IPP-150 buffer (Puig et al., 2001). Pre- cleared cell lysate (3 ml, corresponding to cells from one 225 cm2 flask) was mixed with 7 ml IPP-150 buffer and rotated for 2–4 h at 4 uC in the presence of IgG. The columns were washed with IPP-150 then with TEV-cleavage buffer (IPP-150 supplemented with 1 mM DTT and 0.5 mM EDTA). The beads were transferred to Micro Bio- Spin Chromatography columns (Bio-Rad) and incubated at 4 uC overnight in 300 ml TEV buffer containing TEV-enzyme (30 U). The eluate was collected by low speed centrifugation and dialysed (50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 200 mM NaCl, 10 % glycerol, 1 mM DTT) using a 20 000 MWCO Dialysis Cartridge (Pierce). Journal of General Virology 89