FIG 5.

Prp18 stimulates viral RNA synthesis through interaction with NP. (A) Schematic diagram of Prp18 deletion mutants. a.a, amino acids. (B) Intracellular localization of GST-tagged Prp18 deletion mutants. HeLa cells were transfected with plasmids expressing GST-Prp18, GST-Prp18C1, GST-Prp18C2, and GST-Prp18Δ71. At 24 h posttransfection, cells were subjected to indirect-immunofluorescence assays with anti-GST antibody. DAPI, 4′,6-diamidino-2-phenylindole. (C) GST pulldown assays of GST-Prp18 deletion mutants. HEK293T cells were transfected with plasmids expressing GST-Prp18, GST-Prp18C1, GST-Prp18C2, and GST-Prp18Δ71. At 24 h posttransfection, cells were infected with influenza virus at an MOI of 3. At 6 hpi, cell lysates were prepared and subjected to GST pulldown assays. Coprecipitated proteins were analyzed by Western blotting with anti-PB1, anti-PB2, anti-PA, anti-NP, and anti-GST antibodies. nls, nuclear localization signal. (D) HEK293T cells were transfected with plasmids expressing NP and either GST-Prp18, GST-Prp18C1, GST-Prp18C2, or GST-Prp18Δ71. At 24 h posttransfection, cell lysates were prepared and subjected to a GST pulldown assay. Coprecipitated proteins were analyzed by Western blotting with anti-NP and anti-GST antibodies. (E) Purified recombinant GST, GST-Prp18, GST-Prp18C2, and GST-Prp18Δ71 were separated by 10% SDS-PAGE and visualized by staining with Coomassie brilliant blue. (F) Effect of Prp18 on cell-free RNA synthesis. Cell-free RNA synthesis was carried out with 0.3 pmol (lanes 1, 4, 7, and 10), 1 pmol (lanes 2, 5, 8, and 11), and 3 pmol (lanes 3, 6, 9, and 12) of GST (lanes 1 to 3), GST-Prp18C2 (lanes 4 to 6), GST-Prp18WT (lanes 7 to 9), and GST-Prp18Δ71 (lanes 10 to 12), as described in the legend of Fig. 1.