Figure 1.
Influenza virus increases SK1 activation, which is critical for viral replication.
(A and B) HEK293 (A) or A549 (B) cells were infected with influenza A/WSN/33 virus (FLU) at an MOI of 3 for the indicated times. The levels of pSK1 or SK1 were analyzed by Western blotting. GAPDH or α-tubulin was used as internal loading control. The relative intensities for each band of pSK1 and SK1 were determined based on the control protein expression by densitometery and depicted below each blot. The relative level of protein at 0 hr was set as 1.0. (C and D) MDCK (C) or A549 (D) cells were treated with solvent (DMSO; -), DMS (5 µM), or SKI-II (10 µM) upon influenza virus infection at an MOI of 1. Cell lysates were used for Western blot analysis to detect viral proteins M1, M2, NS1, NS2, NP, and α-tubulin at 12 hpi. (E and F) MDCK cells were treated with solvent (-) or SKI-II upon influenza virus infection at an MOI of 5. The expression of (+) or (-) RNA of viral RNA polymerase subunit PB2 (E) or viral NP (F) was analyzed by real-time quantitative PCR (qPCR) at 5 hpi. Three reactions per sample were carried out. Values are means ± SEM of three reactions per sample. ***p<0.001. (G) MDCK cells were treated with solvent or SKI-II (3, 10, or 30 µM) and infected with influenza virus at an MOI of 0.01. At 24 hpi, a plaque assay was performed to determine virus titer (plaque forming unit, PFU/mL) at each condition. Values are means ± SEM. N = 3/group. ***p<0.001. (H) A549 cells were treated with solvent or SKI-II (10 µM) and infected with influenza virus at an MOI of 0.1. At 12, 36, or 60 hpi, viral titers (PFU/mL) were determined by a plaque assay. Values are means ± SEM (n=3). ***p<0.001. (I and J) SK1-overexpressing HEK293 (I) or A549 (J) cells were transfected with scramble siRNA control (siCTR) or siRNA targeting SK1 (siSK1). After 3 days, the cells were infected with influenza virus at an MOI of 1 (I) or 3 (I and J). At 12 hpi, Western blot analysis was performed to detect pSK1, SK1, viral proteins M2, NS1, NS2, NP, and α-tubulin.
Figure 2.
SK inhibition does not interfere with the viral entry step.
(A and B) MDCK cells were infected with influenza virus (1 MOI) for 1 hr, followed by washing with PBS. Cells were then treated with SKI-II (10 µM) (A) or DMS (5 µM) (B) at 1, 2, 3, or 4 hpi. The expression of viral proteins M1 and M2 was detected by Western blot analysis at 8 hpi. (C and D) MDCK cells were treated with solvent or SKI-II (10 µM) upon influenza virus infection at an MOI of 1. The synthesis of (+) or (-) viral PB2 (C) or NP (D) RNA was analyzed by qPCR at 1 or 2 hpi. The RNA level at 1 hpi was set as 1.0. Values are means ± SEM of three reactions per sample. (E) A549 cells were treated with solvent, DMS (5 µM), or SKI-II (10 µM) and infected with influenza virus at an MOI of 10. The virus was allowed to attach to the cells at 4°C for 1 hr and the cells were incubated at 37°C for an additional 1 hr. Western blot analysis was performed to detect internalized viral M1 and GAPDH. The relative intensities for each band of viral M1 are shown.
Figure 3.
SK inhibition suppresses influenza virus replication by impairing activation of the NF-κB signaling pathway.
(A) MDCK cells were left untreated or treated with BAY11-7082 (2.5 µM) and infected with influenza virus at an MOI of 1. The expression of viral proteins M1, NS1, NS2, and actin was assessed by Western blotting at 7 hpi. (B) MDCK cells were treated with solvent or SKI-II (10 µM) and uninfected (Mock) or infected with influenza virus at an MOI of 3. At 0.5, 1, 2, or 4 hpi, Western blot analysis was performed to detect pIKKαβ, IKKαβ, and α-tubulin. (C) MDCK cells were treated with solvent alone or SKI-II (10 µM) and uninfected (Mock) or infected with influenza virus (3 MOI). At 3, 4, 5, or 6 hpi, Western blotting was performed to detect p-p65, p65, and viral M1. (D) MDCK cells were mock-infected or infected with influenza virus at an MOI of 3. They were fixed, permeabilized, and stained with antibodies against NF-κB subunit p65 (red) at 4 hpi and DRAQ5 dye to detect nuclei (blue). Representative confocal images are shown. Scale bar = 50 µm. (E) MDCK cells were co-transfected with NF-κB luciferase reporter plasmid and control Renilla luciferase plasmid. After 12 hrs, cells were treated with solvent alone or SKI-II (10 µM) upon influenza A/Hong Kong/8/68 virus infection at 5 MOI for 7 hrs or were transfected with influenza viral RNA (100 ng) for 7 hrs. Cell lysates were analyzed for luciferase activity with a luminometer. Relative luciferase activities are shown. Values are means ± SEM (n=3). *p<0.05.
Figure 4.
SK inhibitor treatment interferes with nuclear export of influenza viral RNP complex.
(A) MDCK cells were treated with solvent (-), DMS (5 µM), or SKI-II (10 µM), or leptomycin B (LMB, 10 ng/mL) and infected with influenza virus at 1 MOI for 12 hrs. Cells were then fixed, permeabilized, and stained with antibodies against viral NP (green) and with DRAQ5 dye to detect nuclei (red). (B and C) MDCK cells were treated with solvent, DMS (5 µM), SKI-II (10 µM), or LMB (10 ng/mL) and infected with influenza virus at an MOI of 1 for 12 hrs. After staining with antibodies against viral M1 (B) or NS1 (C), viral proteins (green) were visualized by confocal microscopic analysis. DRAQ5 dye was used to stain nuclei (red). (D) MDCK cells were treated with solvent or SKI-II (10 µM) and infected with influenza A/Hong Kong/8/68 (FLU-HK) virus at 1 MOI for 10 hrs. (E) MDCK cells were infected with influenza virus at an MOI of 1 and then treated with cycloheximide (CHX) at 6 hpi or left untreated. The cells were then treated with solvent or SKI-II at 6.5 hpi for an additional 4.5 hrs (top panel). For viral NP and α-tubulin detection, Western blot analysis was performed (middle panel). Viral NP expression was analyzed by confocal microscopy (bottom panel). Cells were stained with DRAQ5 dye to detect nuclei. Scale bar = 50 µm.
Figure 5.
SK inhibition impairs virus-induced activation of ERK/p90RSK/AKT to inhibit RanBP3-mediated nuclear export of viral NP.
(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.