We therefore first tested the ability of the mutant PB1 polypeptides to support viral gene expression in 'minireplicon' assays (26, 35) . To reconstitute active viral RNPs, plasmids encoding the three influenza A virus polymerase proteins and nucleoprotein were co-transfected with a further plasmid that expressed a synthetic vRNA molecule encoding luciferase in antisense from an RNA polymerase I promoter. The luciferase levels in transfected cells therefore represent a measure of the transcriptional activity of the polymerase complex. When luciferase values were normalized to those obtained with WT PB1, all of the segment 2 mutants with unaltered PB1 sequences gave values that fluctuated around the 100% mark (between 62 and 129% of normal), while a sample from cells lacking PB1 gave <1% output ( Figure 3A ). However, three of the four non-synonymous changes in PB1 were deleterious to transcriptional activity. The T32G (V3G) and A78T+G101T (G26V) polymerases were the most impaired, with luciferase readings of 13 and 20% of WT respectively. The G28A mutant (D2N) was marginally impaired, producing luciferase activity at 54% of WT. Only the C74G mutant (A17G, a relatively conservative change) supported transcriptional activity in the range of that observed with the mutants with synonymous changes in PB1 (72%). The reduced ability of the T32G and A78T+G101T mutants to support virus gene expression was also seen when authentic segment 7 was used as a RNP substrate and M1 and M2 accumulation analysed by western blot (data not shown) or when unspliced segment 7 mRNA accumulation was measured by reverse transcriptase-primer extension assay ( Figure 3B ).