Plasmids pcDNA-PB2, -PA and -NP, containing cDNA copies of the influenza A/PR/8/34 (PR8) genes as well as plasmid pPolI-Flu-ffLuc containing an influenza virus-based luciferase minireplicon vRNA under the control of the human RNA polymerase I (Pol I) promoter have been previously described (7, 26) . Dual promoter reverse genetics plasmids for PR8 segments 1 and 3-8 and a pPol-I segment 7 clone were donated by Professor Ron Fouchier (27) . A similar construct for segment 2 cloned from the NIBSC strain of PR8 is described in (7) . To assess PB1-F2 expression in vitro, a CAT fragment was ligated into SmaI/XbaI digested pcDNA-PB1 in frames 1, 2 or 3. To analyse viral gene expression from transfected plasmids, nucleotides 1-380 of EF467819 were subcloned into pEGFPN1 (Clontech) as a AgeI/KpnI fragment. Site directed mutagenesis was then employed to position the green fluorescent protein (GFP) ORF into frame with either the PB1 or PB1-F2 reading frames while concurrently removing the GFP AUG codon. Additional segment 2 mutations as detailed in the results section were made using site directed mutagenesis with the wild-type segment plasmids as templates. For brevity, the sequences of the mutational oligonucleotides are not given but are available on request. All plasmids were sequence verified.