Figure 2.
IAV multiplication is impaired in ERI1-depleted cells and ERI1 interacts with viral proteins. (A, B) A549 cells were treated with control (black) or single ERI1 siRNA (gray, ERI1#1) and infected with the following viruses at the indicated moi in pfu/cell: A/WSN/33(H1N1) (WSN, moi 10−4); A/Paris/650/2004(H1N1) (sH1N1, moi 10−3); A/Bretagne/7608/2009(H1N1pdm09) (pH1N1, moi 10−4), A/Centre/1003/2012(H3N2) (H3N2, moi 10−3). At 24 hpi (A), or at several time points post-infection (18, 24, 36, 48, 72 hpi) (B), viral titers were determined by plaque assay on MDCK-SIAT cells. The results are expressed as the mean ± SEM of triplicates and the significance was tested with a multiple t test, using the Holm-Sidak method, in GraphPad Prism software (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Interaction of ERI1 with sH1N1 and pH1N1 viral proteins was tested by applying the NLR (normalized luminescence ratio) method that takes into account the background noise of interaction of the Gluc1 or Gluc2 fusion partners. Positive threshold values, calculated as in (38), are represented by hatched bars. The results are expressed as the mean ± SEM of triplicates. (D) HEK-293T cells were co-transfected with Strep-ERI1 or Strep-mCherry (as a control), and Gluc1-PB2-WSN, -PB1-WSN, -PA-WSN, -NP-WSN or –M1-WSN. At 24 hpt, Strep tagged proteins were purified with sepharose Strep-Tactin beads. PA, found as a non-interacting partner of ERI1 in our GPCA screen was used as a control of non-specific interaction. Strep-tagged eluates were analyzed by immunoblot to detect Strep and Gluc1 tagged proteins. Results representative of three independent experiments are shown. (E) HEK-293T cells were co-transfected with Strep-ERI1 or Strep-mCherry (as a control) along with untagged PB2, PB1 and PA. At 24 hpt, Strep tagged proteins were purified with sepharose Strep-Tactin beads. Strep-tagged eluates were analyzed by immunoblot to detect Strep and the viral proteins. Results representative of three independent experiments are shown.