Influenza A Virus NS1 interacts with mouse Riplet.

(A) At 30 h posttransfection with Myc-tagged mouse Riplet (Myc-mRiplet) together with NS1-PR8, HEK293T cells were mock-treated or infected with SeV (10 HA units/ml) for 10 h. V5-tagged hTRIM25 was co-transfected as positive control. WCLs were subjected to IP with anti-Myc (Riplet) or anti-V5 (TRIM25), followed by IB with anti-NS1 antibody. (B) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies. (C) Localization of NS1-PR8 and mouse Riplet in HeLa cells determined by confocal microscopy. At 30 h posttransfection with Myc-mRiplet alone, NS1-PR8 alone, or NS1 together with Myc-mRiplet, HeLa cells were stained with anti-Myc (green), anti-NS1 (red), and DAPI (nucleus, blue). Fifty cells from three independent experiments were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test. (D and E) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus expressing NS1 PR8, Cal04, HK156, Tx91, SwTx98, or Pan99 at an MOI of 2 (D), or R38A/K41A or E96A/E97A NS1 mutant at an MOI of 4 (E). 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies.