B. G. Bradel-Tretheway and others Fig. 1. Subcellular distribution and activity of purified, adenovirus-expressed WSN polymer- ase. (a) A549 cells were transduced with rAd5-PAWSN-TAP alone or with a combination of rAd5-PAWSN-TAP, -PB1WSN and -PB2WSN (W/W/W). Nuclear and cytoplasmic extracts were analysed by immunoblotting using PA-, PB1- or PB2-specific antibodies. The effec- tiveness of the subcellular fractionation method was confirmed by probing against the cytoplasmic proteins, actin and b-tubulin as well as against RanBP5 (which is found both in the cytoplasm and nucleus). NS, non- specific protein band detected when probing against PB1/2. Cells transduced with an adenovirus vector expressing LacZ served as a negative control. Twofold more cell equiva- lents were loaded for the nuclear extracts than the cytoplasmic extracts. (b) IgG-purified nuclear and cytoplasmic extracts were ana- lysed by silver staining prior to (c) comparison with total cell lysate in an ApG-primed transcription assay (1 h at 30 6C). Extracts from equivalent numbers of transduced A549 cells were used. (d) IgG-purified extracts from total cellular lysates were analysed by silver staining (upper panel) and immunoblotting against RanBP5 (lower panel). The positions of PA, PB1 and PB2 are shown on the right. the intact 3P complex (unless otherwise indicated). For all following experiments 3P complexes purified from total cellular lysates were utilized. The 3P complex from the A/Viet/1203/04 (VN1203) strain is highly active, and both the WSN and VN1203 complexes are competent for cap- independent and cap-dependent transcription We purified 3P complexes (PA/PB1/PB2) from the VN1203 (H5N1) (H/H/H) and the A/WSN/33 (H1N1) (W/W/W) strains as well as complexes in which the PB2 subunits were exchanged (H/H/W, W/W/H) or omitted (H/H). The purified complexes were analysed by silver staining and immunoblotting against PB2 (Fig. 2a top and bottom panel, respectively). All complexes, except the negative control (H/H), were able to initiate transcription at 30 uC in a cap-independent fashion by utilizing ApG as a primer (Fig. 2b, top panel). A fully extended primer that yielded a 14 nt transcript was detected for all complexes. An additional band above the 14 nt extension product is likely due to some terminal transferase ability of the IAV polymerase. Influenza viruses use a cap-snatching mechanism to initiate transcription, which involves recognition and binding of the cap structure of host mRNAs by PB2, followed by 2926 cleavage of the capped mRNA at nucleotide 10–14 by the endonucleolytic activity of PB1 (Guilligay et al., 2008; Li et al., 2001; Plotch et al., 1981). These short RNAs are then utilized by the polymerase as primers to initiate transcrip- tion. We expected that cleavage of the provided cap-donor by the 3P complex would result in the generation of short RNAs able to bind to the 39-vRNA template (14 nt). The resulting transcription product (cap primer +14 nt) was detected as a triplet band in our assays (Fig. 2b, bottom panel), which could be due to multiple cleavage sites in the primer or to residual nuclease activity (Fodor et al., 2002). All purified complexes were able to utilize rabbit globin mRNA to prime transcription. Thus, all of the complexes, except the negative control (H/H), were competent for cap-binding and endonucleolytic activity. Only a fully assembled, trimeric complex is transcriptionally active (Deng et al., 2005; Perales & Ortin, 1997). Relative levels of transcriptional activity for the various complexes were measured by normalizing to the levels of PB2 (Fig. 2a, bottom panel). This analysis revealed that the VN1203 3P complex was 40-fold more active in the ApG- primed (Fig. 2c) and about 30-fold more active in the cap- primed transcription assay (Fig. 2d) than the WSN 3P complex, and that the PB2 subunit contributes to this enhanced activity. These findings are consistent with those reported by Taubenberger, who noted that IAV polymerase Journal of General Virology 89