SK inhibition impairs virus-induced activation of ERK/p90RSK/AKT to inhibit RanBP3-mediated nuclear export of viral NP.

<p>(A and B) HEK293 cells were transfected with scramble siRNA (siCTR) or siRNA targeting RanBP3 (siRanBP3); then the cells were infected with influenza virus at an MOI of 0.01 (A) or 1 (B). RanBP3, pRanBP3, viral M1, and α-tubulin were detected by Western blotting at 30 hpi (A). Viral NP (green) and nuclei (DRAQ5 dye, red) were visualized by confocal microscopy at 12 hpi (B). (C and D) MDCK cells were treated with solvent, DMS (5 µM), or SKI-II (10 µM) and infected with influenza virus at an MOI of 5 for the indicated times (C) or 6 hrs (D). Cell lysates were used for Western blot analysis to detect pRanBP3, RanBP3, α-tubulin, or actin. (E) MDCK cells were left untreated or treated with U0126 (10 µM) or LY294002 (10 µM) and infected with influenza virus at 3 MOI for 6 hrs. Western blot analysis was performed to detect pRanBP3, RanBP3, and GAPDH. (F) MDCK cells were treated with solvent alone or SKI-II (10 µM) and infected with influenza virus at an MOI of 1. Western blot analysis was performed to detect pERK, ERK, p-p90RSK, p90RSK, pRANBP3, or α-tubulin at 12 hpi. (G) MDCK cells were treated with solvent or SKI-II (10 µM) and mock-infected or infected with influenza virus at an MOI of 3. At 8 hpi, pAKT, AKT, and α-tubulin were detected by Western blot analysis.