Fig 5.

NS1 can downregulate RhoA activity by direct interaction with RhoA protein. (A) Transfected A549 cell lysates were incubated with equal amounts of GST-Rhotekin bound to glutathione-Sepharose 4B beads. After washing, the bound proteins were analyzed by Western blotting with an anti-RhoA antibody. The data represent the mean fold changes ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (B) A549 cells were mock infected (mock) or infected with virus for 24 h, and then the cells were collected and analyzed by Western blotting with antibodies as indicated. At the same time, the cell lysates were incubated with equal amounts of GST-Rhotekin bound to glutathione-Sepharose 4B beads. After washing, the bound proteins were analyzed by Western blotting with an anti-RhoA antibody. The data are shown as means ± the SD of three independent experiments (*, P < 0.05 relative to the control group). (C) Whole 293T cell lysates transfected with Myc-tagged RhoA were subjected to GST pulldown, followed by immunoblotting (IB) with the Myc antibody. CBB, Coomassie brilliant blue staining. (D) The 293T cells were cotransfected with Myc-tagged RhoA and either pcDNA3.0-TRAF6 or pcDNA3.0-NS1 and with Flag-tagged NS1 and either pcDNA4.0 or pcDNA4.0-RhoA. Flag antibodies were used with the cell lysates for immunoprecipitation (IP), followed by Western blotting (WB) with Myc, or for immunoprecipitation with Myc, followed by Western blotting with Flag. (E) A549 cells were transfected with pEGFP-N1 and pEGFPN1-NS1 for 24 h and then fixed, permeabilized, and stained for RhoA (red). Yellow indicates overlap. Scale bar, 10 μm. (F) The 293T cells were transfected with Flag-tagged TRAF6 and Flag-tagged NS1, separately. Then, the cell nuclear lysates were subjected to immunoprecipitation with Flag antibody, followed by Western blotting with RhoA antibody. (G) A549 cells were mock infected (mock) or infected with virus for 24 h, and the cells were subjected to immunoprecipitation with NS1 antibody, followed by Western blotting with RhoA. (H) Whole 293T cell lysates transfected with Myc-tagged RhoA97 and RhoA194 were subjected to GST pulldown, followed by immunoblotting with Myc antibody. (I) Whole 293T cell lysates transfected with Myc-tagged RhoAS188A and RhoAS188E were subjected to GST pulldown, followed by immunoblotting with Myc antibody. (J) Myc-tagged RhoA-transfected 293T cell lysates were incubated with equal amounts of GST, GST-NS1-R (N terminal, 1 to 73 aa), or GST-NS1-E (C terminal, 73 to 230 aa). The bound proteins were analyzed using Myc antibody.