Fig 4.

In the absence of transcription/replication, PB2 mutations do not affect the amount of PB2 precipitated by NP. (A) PB1 mutation D446Y inhibits 50-92 polymerase activity. 293T cells were transfected with pCAGGS 50-92 NP, FLAG-tagged PB2 WT or 627K, PA, and PB1 WT or PB1 D446Y as well as a virus-like firefly luciferase reporter plasmid and pCAGGS Renilla. Firefly luciferase production was measured 12 h posttransfection. Values were normalized to Renilla expression. Results shown are the means ± standard deviations from three independent experiments. RLU, relative light units. (B) 293T cells were transfected with pCAGGS 50-92 NP, PB1 D446Y, PA, and WT or mutated PB2-FLAG and a virus-like firefly luciferase reporter plasmid. At 12 h posttransfection, cell lysates were prepared and subjected to NP immunoprecipitation prior to Western blot analysis. Results are from one experiment representative of three independent experiments. (C) The levels of coimmunoprecipitated PB2 in panel A were normalized to precipitated NP using ImageJ.