Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication
Fig 6
PRRSV infection induces ER stress and SOCE channel take up extracellular calcium.
(A-C) Marc-145 cells were infected with PRRSV (MOI = 0.1) or treated with tunicamycin for 24 h. (A) Cell lysates were analyzed by western blotting for STIM1, Orai1, GRP78, CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (B) Cytoplasmic Ca2+ and (C) ER Ca2+ were determined by fluorescence of Fluo-8. (D) Marc-145 cells were transfected with mCherry-STIM1 and GFP-Orai1 for 24 hours, followed by infection with PRRSV. mCherry-STIM1 and GFP-Orai1 were visualized by confocal microscopy at indicated timepoints. Scale bar, 5 μm. (E-I) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with 4-PBA (2.0 mM) or DMSO for 24 h. (E) Cell lysates were analyzed by western blotting for STIM1, Orai1, GRP78, CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (F) Cytoplasmic Ca2+ and (G) ER Ca2+ were determined by fluorescence of Fluo-8. (H) TCID50 of PRRSV in cell supernatants. (I) The effect of 4-PBA on Marc-145 cell viability. Marc-145 cells were treated with 4-PBA at indicated concentrations or DMSO for 24 h, and then analyzed with CCK-8 system. (J-N) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with ML-9 HCL (25 μM) or DMSO for 24 h. (J) Cell lysates were analyzed by western blotting for STIM1, Orai1, GRP78, CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (K) Cytoplasmic Ca2+ and (L) ER Ca2+ were determined. (M) TCID50 of PRRSV in cell supernatants. (N) The effect of ML-9 HCL on Marc-145 cell viability. Marc-145 cells were treated with ML-9 HCL at indicated concentrations or DMSO for 24 h, and then analyzed with CCK-8 system. (O-T) STIM1 is essential for PRRSV-induced Ca2+ influx, activation of CaMKII-AMPK-mTOR-LC3II signaling, and PRRSV efficient replication. (O) Western blotting was used to quantitate the level of STIM1 in siRNA1-, siRNA2-, siRNA3-, Mix (the equal amounts of siRNA1, siRNA2 and siRNA3) or siNC-transfected Marc-145 cells. (P) The cell viability of Marc-145 cells transfected with in siRNA1, siRNA2, siRNA3 or Mix (the equal amounts of siRNA1, siRNA2 and siRNA3) targeting to STIM1, or siNC. (Q-T) Marc-145 cells were transfected with siRNA Mix (the equal amounts of siRNA1, siRNA2 and siRNA3) targeting to STIM1 or siNC for 24 h, and mock or infected with PRRRV (MOI = 0.1) for another 24 h. (Q) Cell lysates were prepared and analyzed by immunoblotting using anti-STIM1, anti-Orai1, anti-GRP78, anti-CaMKII, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-LC3-I/II, anti PRRSV-N, and anti-β-actin antibodies. (R) Cytoplasmic Ca2+ and (S) ER Ca2+ were determined. (T) TCID50 of PRRSV in cell supernatants. The protein levels were quantified by Image J and normalized to β-actin. The data are representative of results from three independent experiments. Error bars indicate the mean (± SD) of three repeats. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.