Silver staining and immunoblotting. Proteins were heat-denatured and separated by 7.5 % SDS-PAGE prior to immunoblotting. For silver staining, gels were treated according to the manufacturer’s protocol (SilverXpress Silver Staining kit, Invitrogen). Antibodies used were (Santa Cruz): anti-a-tubulin (mouse, TU-02), anti-PB1 (vK-20), anti-PB2 (vN-19), anti-actin (H-196) and anti-goat IgG HRP (Sc-2033). ECL anti-rabbit IgG HRP (NA934V) and ECL anti-mouse IgG HRP (NA931V) were purchased from Amersham and anti-Penta-His (mouse) from Qiagen. ApG- and globin-primed transcription assay. The ApG-primed transcription assay was performed basically as described elsewhere (Fodor et al., 2002). The amount of purified 3P complex used in transcription assays was normalized either in terms of PB2 content (as measured by immunoblotting) or in terms of transcriptional activity at 30 uC. IgG-purified, normalized samples were mixed with transcription buffer (25 mM Tris-Cl, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.25 Nonidet P-40, 12.5 % glycerol) supplemented with 0.25 U RNasin ml21, 1 mM each ATP/ UTP/CTP, 0.1 mM GTP, 0.15 mM [a-32P]GTP (3000 Ci mmol21), 1.6 mM each 59- (59-AGUAGAAACAAGGCC-39) and 39-end vRNA (59-GGCCUGCUUUUGCU-39) and 0.3 mM ApG (Biosynthesis). The reaction volume (5 ml) contained 2 ml normalized protein sample. Transcription was allowed to occur for 1 h at 30, 34, 37, 39 or 42 uC and the reaction was terminated by the addition of 4 ml Stop-buffer (10 mM EDTA pH 8.0, 90 % formamide). Transcription products were separated by 18 % PAGE in 7 M urea and detected by autoradiography using a PhosphorImager (Molecular Dynamics). Densitometry was performed using OptiQuant (Version 3.10, Packard Instrument). The globin-primed (cap-dependent) transcription assay was per- formed similarly. Instead of ApG rabbit-globin mRNA (Sigma) was used as a cap-donor (10 ng per 5 ml reaction volume) and the incubation time was 90 min (Hara et al., 2006). Thermo-stability assay. Equivalent amounts of polymerase in 2 ml (as determined by polymerase activity at 30 uC) were pre-incubated at 30 uC for 5, 10, 15, 20, 25, 30, 60 and 120 min. After pre-incubation 3 ml ApG-containing transcription buffer, supplemented as described above, was added to the sample and transcription was allowed to occur at 30 uC for 1 h. RESULTS Adenoviral expression of the IAV polymerase in human lung epithelial cells (A549) A549 cells were transduced with either a LacZ-expressing control adenovirus, a PAWSN-TAP-expressing virus or a combination of three viruses expressing PAWSN-TAP, PB1WSN and PB2WSN (W/W/W). To test whether the adenovirus-mediated expression system might alter the normal distribution of the IAV 3P complex, we analysed the cellular distribution of PA in the absence or presence of PB1 and PB2. To do this, nuclear and cytoplasmic extracts were prepared from vector-transduced A549 cells, sepa- rated by SDS-PAGE and blotted with antibodies directed against PA, PB1 and PB2. To confirm the effectiveness of our subcellular fractiona- tion method, we also examined the presence of a-tubulin and actin (both cytoplasmic proteins), and of RanBP5 http://vir.sgmjournals.org Role of PB2 in IAV temperature sensitivity (which is found predominantly in the cytoplasm, but also in the nucleus) (Deane et al., 1997). As shown in Fig. 1(a), nuclear fractions were lacking in both a-tubulin and actin, but contained RanBP5. Fig. 1(a) also shows that PA, when expressed alone, mainly accumulated in the cytoplasm with less than 10 % in the nucleus; in contrast, when co- expressed with PB1 and PB2 (W/W/W), PA was readily detected in the nucleus (about 30 %), as expected (Fodor & Smith, 2004; Nieto et al., 1992). Thus, the adenovirus expression system had no detectable effect on the expected subcellular localization of the IAV 3P complex and its individual component proteins. Transcriptional activity of the WSN 3P complex purified from A549 cells The polymerase complex was purified from whole-cell lysates, nuclear and cytoplasmic extracts by binding PA- TAP via its protein A domain to IgG-beads. All proteins that interact with PA, including PB1, PB2 and host factors binding to the 3P complex, were expected to be pulled down by this approach. The complex was released from the beads by cleavage with TEV-enzyme and the purity of the complex was analysed by silver staining (Fig. 1b, d). We next tested the transcriptional activity of the purified polymerase complexes using an ApG-primed transcription assay in which the 39- and 59-vRNA and the dinucleotide ApG were provided. In this assay, the 3P complex initiates extension of the bound dinucleotide; the fully extended product of this reaction is a 14 nt RNA fragment. No extension products were detected when the ApG-primed transcriptional assay was performed with IgG-purified extracts from cells expressing LacZ or PA alone. Only the fully assembled trimeric polymerase complex (W/W/W) generated an extension product (Fig. 1c). We did not observe any difference in transcriptional activity of purified polymerase complexes from total cell lysates, or from either nuclear or cytoplasmic extracts (Fig. 1c); the apparently lower transcriptional activity of the nuclear polymerase complex can be attributed simply to the lower amount (~30 % less) of assembled 3P complex present in the nucleus (Fig. 1b). For further biochemical studies, 3P complexes were isolated in larger quantities from total cell lysates and analysed by silver staining (Fig. 1d, top panel). The most abundant protein in the purified complex was PA, which was precipitated as a monomer, or complexed with either PB1 (PA/PB1) or PB1 and PB2 (PA/PB1/PB2). The second most abundant protein was PB1, which was pulled down as a dimeric (PA/PB1) or trimeric (PA/PB1/ PB2) complex. The presence of dimeric (PA/PB1) complex can be inferred by the presence of the host-factor RanBP5 (Fig. 1d, bottom panel; Supplementary Fig. S1, available in JGV Online) that is known to interact with PB1 or PB1/PA (Deng et al., 2006). PB2 was the least abundant protein since it was only precipitated in the context of the intact 3P complex (PA/PB1/PB2); therefore in our future experi- ments, PB2 content was used to normalize overall levels of 2925