FIG 6.
The ANP32-polymerase interaction is stabilized at RNPs. (a to c) HEK 293T cells were transfected with expression plasmids encoding PB2 627E, PA, chANP32Aluc2, PB1 D446Yluc1 (a) or PB1luc1 (b and c), and 0, 10, 100, 200, 300, or 400 ng of either a PolI plasmid expressing an influenza virus-like vRNA of 76 nt in length (a), a PolI plasmid expressing an influenza virus-like vRNA of 1,723 nt in length (b), or a PolI plasmid expressing an influenza virus-like cRNA of 1,723 nt in length (c). Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Western blotting assay to show expression of tagged plasmids is shown beneath the graphs. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. (d and e) Cells were transfected with PB1 D446Yluc1, huANP32Aluc2, PA, 400 ng of a PolI plasmid expressing a 1,723-nt vRNA or cRNA, and either PB2 627K (d) or PB2 627E (e). Statistical significance was assessed by one-way analysis of variance, and comparisons were made to sample with no added RNA (left black bar). ns, nonsignificant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Results representative of three independent experiments.