Fig 1.
Infection with PRRSV results in increased cytoplasmic Ca2+.
(A-C) Marc-145 cells were infected at MOI of 0.1 for different timepoints (6, 12, 24, 36 h). (A) PRRSV-GFP growth kinetics. (B) Increase in cytoplasmic Ca2+ over infection time course. (C) The fluorescence of Ca2+ (Rhod-2, Red) was observed using confocal microscopy after PRRSV-GFP infection at different timepoints. Scale bar, 10 μm. (D-F) Marc-145 cells were infected with PRRSV-GFP at different MOIs for 24 h. (D) Viral titers as a function of MOI at 24 hpi. (E) Cytoplasmic Ca2+ as a function of MOI. (F) The fluorescence of Ca2+ (Rhod-2, Red) was observed using confocal microscopy after PRRSV-GFP infection in different MOIs. Scale bar, 10 μm. Data are expressed as means ± SD (n = 3). *p<0.05; **p < 0.01; ***p < 0.001.
Fig 2.
Intracellular Ca2+ benefits PRRSV replication.
(A-E) BAPTA inhibits PRRSV infection. Marc-145 cells were mock or PRRSV infected (MOI = 0.1) for 24 h with different doses of BAPTA-AM. (A) RT-PCR for detection of PRRSV ORF7 (N protein). (B) Western blot of PRSSV N protein. (C) Viral titers as a function of BAPTA concentration. (D) Cytoplasmic Ca2+ induction after PRRSV infection, with and without BAPTA-AM. (E) Cell viability of Marc-145 cells treated with BAPTA-AM at indicated concentrations or DMSO for 24 h. (F-J) Exogenously supplied Ca2+ promotes PRRSV replication. Marc-145 cells were infected with PRRSV (MOI = 0.1) and then cultured in normal (Ca2+ = 1.80 mM) or Ca2+-free medium for 24 h. (F) RT-PCR for detection of PRRSV ORF7 (N protein). (G) Western blot of PRSSV N protein. (H) Viral titers as a function of exogenous Ca2+. (I) Cytoplasmic Ca2+ in cells after PRRSV infection in normal or Ca2+-free medium. (J) Cell viability of Marc-145 cells cultured with Ca2+-free or normal medium for 24 h. The protein levels were quantified by Image J and normalized to β-actin. Data are expressed as means ± SD. *p<0.05; **p < 0.01; ***p < 0.001. The experimental data are representative of results from three independent experiments.
Fig 3.
Cytosolic Ca2+ promotes PRRSV replication through autophagy.
(A-C) BAPTA-AM inhibits PRRSV induced autophagy. Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with BAPTA-AM for 24 h. (A) The ratio of endogenous LC3I to LC3II determined by western blotting. (B) Marc-145 cells were transfected with p-mCherry-GFP-LC3 for 24 h and then infected with PRRSV (MOI = 0.1) and treated with BAPTA-AM for another 24 h. LC3 puncta were visualized by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bar = 5 μm. (C) Quantitation of LC3 puncta formation. Results represent the number of LC3 puncta per cell in panel B (n = 20). (D-F) Extracellular Ca2+ promotes PRRSV induced autophagy. (D) Marc-145 cells were infected with PRRSV (MOI = 0.1) and cultured in normal or Ca2+-free medium for 24 h. The ratio of endogenous LC3I to LC3II determined by western blotting. (E) LC3 puncta were visualized by confocal microscopy. Scale bar, 5 μm. (F) Quantitation of LC3 puncta formation. Results represent the number of LC3 puncta per cell in panel E (n = 20). (G) The transmission electron microscopy analysis of virus-infected cells. Marc-145 cells were treated as shown in different groups (DMSO, DMSO + PRRSV, BAPTA-AM + PRRSV, Ca2+-free medium, Ca2+-free medium + PRRSV). 24 h later, cells were fixed and processed for electron microscopy analysis. A indicated the single- and double-membrane vesicles of autophagosome. The numbers of autophagosome were quantified from 20 different images in each group. (H-I) Marc-145 cells were transfected with control siRNA (siNC) or siRNA targeting ATG7 (siATG7) for 24 h, and then infected with PRRSV (MOI = 0.1) for another 24 h in normal or Ca2+-free medium. (H) PRRSV-N protein levels and the LC3-I/II ratio was determined by western blotting. (I) TCID50 of PRRSV in cell supernatants. The protein levels were quantified by Image J and normalized to β-actin. Data are expressed as means ± SD, n = 3 in G or n = 20 in C, F and G. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 4.
PRRSV induces autophagy through CaMKII-AMPK-mTOR pathway.
(A) Marc-145 cells were mock or infected with PRRSV (MOI = 0.1) for 24 h. Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (B-E) CaMKII mediated autophagy is essential for PRRSV efficient replication. (B) Western blotting was used to quantitate the level of CaMKII in siRNA1-, siRNA2-, siRNA3-, Mix (the equal amounts of siRNA1, siRNA2 and siRNA3) or siNC-transfected Marc-145 cells. (C) 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 CaMKII, or siNC. (D-E) Marc-145 cells were transfected with siRNA Mix (the equal amounts of siRNA1, siRNA2 and siRNA3) targeting to CaMKII or siNC for 24 h, and mock or infected with PRRRV (MOI = 0.1) for another 24 h. (D) Cell lysates were prepared and analyzed by immunoblotting using anti-CaMKII, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-LC3-I/II, anti PRRSV-N, and anti-β-actin antibodies. (E) TCID50 of PRRSV in cell supernatants. (F-H) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with KN-93 (10 μM) or DMSO treatment for 24 h. (F) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (G) TCID50 of PRRSV in cell supernatants. (H) The effect of KN-93 on Marc-145 cell viability. Marc-145 cells were treated with KN-93 at indicated concentrations for 24 h, and then analyzed with CCK-8 system. (I-K) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with Compound C (10 μM) or DMSO for 24 h. (I) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (J) TCID50 of PRRSV in cell supernatants. (K) The effect of Compound C on Marc-145 cell viability. Marc-145 cells were treated with Compound C at indicated concentrations for 24 h, and then analyzed with CCK-8 system. (L) Marc-145 cells were infected with PRRSV (MOI = 0.1) with or without BAPTA-AM (30 μM) for 24 h. Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. 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 independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Fig 5.
The ER takes up extracellular calcium during PRRSV infection.
(A-C) Marc-145 cells were infected with PRRSV (MOI = 0.1) and cultured in media containing 0, 0.9, 1.8, or 3.6 mM calcium chloride for 24 h. (A) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (B) TCID50 of PRRSV in cell supernatants. (C) Cytoplasmic Ca2+ levels were determined by fluorescence of Fluo-8. (D) ER associated Ca2+ levels were determined by a Fluo-8 calcium flux analysis kit as described in Material and Methods. 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 independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
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.
Fig 7.
ER associated calcium is released into the cytoplasm from IP3R but not RyR channel.
(A-E) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with 10 μM 2-APB or DMSO for 24 h. (A) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (B) Cytoplasmic Ca2+ levels and (C) ER Ca2+ levels were determined by fluorescence of Fluo-8. (D) TCID50 of PRRSV in cell supernatants. (E) The effect of 2-APB on Marc-145 cell viability. Marc-145 cells were treated with 2-APB at indicated concentrations or DMSO for 24 h, then analyzed with CCK-8 system. (F-J) Marc-145 cells were infected with PRRSV (MOI = 0.1) and treated with 50 μM dantrolene sodium or DMSO for 24 h. (F) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (G) Cytoplasmic Ca2+ levels and (H) ER Ca2+ levels were determined by fluorescence of Fluo-8. (I) TCID50 of PRRSV in cell supernatants. (J) The effect of Dantrolene sodium on Marc-145 cell viability. Marc-145 cells were treated with Dantrolene sodium at indicated concentrations or DMSO for 24 h, then analyzed with CCK-8 system. 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.
Fig 8.
PRRSV Nsp2 interacts with STIM1 and GRP78, induces autophagy.
(A and B) The effect of viral non-structural and structural proteins on ER stress. Marc-145 cells were transfected with plasmids encoded non-structural (A) and structural (B) proteins for 24 h. Cell lysates were harvested for western blotting with antibodies against Flag, GRP78 and β-actin. (C-H) PRRSV Nsp2 induces ER stress and autophagy. (C) Marc-145 cells were transfected with pCI-Nsp2-flag or empty vector (pCI-neo) for 24 h then cell lysates were analyzed by western blotting for Flag, STIM1, GRP78, LC3-I/II, and β-actin. (D-E) Marc-145 cells were transfected with pCI-neo or increasing doses of pCI-Nsp2-flag for 24 h. (D) Cytoplasmic Ca2+ and (E) ER Ca2+ levels were determined by fluorescence of Fluo-8. (F) Marc-145 cells were transfected with pCI-Nsp2-flag or pCI-neo together with GFP-LC3 for 24 h. Cells were stained for Flag, nuclei were stained with DAPI, and cells were observed by confocal microscopy. The numbers of GFP-LC3 dots were quantified from 20 different cells in each group. (G) Colocalization of Nsp2 and GRP78 at ER. Cells were stained for Flag, GRP78, Calnexin (ER marker) and nuclei were stained with DAPI; cells were observed by confocal microscopy. Scale bar, 5 μm. (H) Colocalization of Nsp2 and STIM1 at ER. Cells were stained for Flag, STIM1, Calnexin (ER marker) and nuclei were stained with DAPI; cells were observed by confocal microscopy. Scale bar, 5 μm. (I) PRRSV Nsp2 is associated with GRP78 and STIM1. Marc-145 cells were transfected with pCI-Nsp2-flag, pCI-Nsp1α-flag, pCI-GP5-Flag or pCI-neo for 24 h. Cell lysates were immunoprecipitated with anti-Flag antibody, and then subjected to western blot analysis for Flag, STIM1 GRP78, and β-actin. 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.
Fig 9.
Ca2+ promotes PRRSV replication in pulmonary alveolar macrophages (PAMs).
(A-C) PAMs were infected with PRRSV (MOI = 0.1) for 24 h. (A) Cell lysates were analyzed by western blotting for CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (B) Cytoplasmic and (C) ER Ca2+ levels were determined by fluorescence of Fluo-8. (D-G) PAMs were infected with PRRSV (MOI = 0.1) and treated with 25 μM ML-9 HCL or DMSO for 24 h. (D) Cell lysates were analyzed by western blotting for STIM1, Orai1, CaMKII, p-AMPK, AMPK, p-mTOR, mTOR, LC3-I/II, PRRSV-N, and β-actin. (E) Cytoplasmic and (F) ER Ca2+ levels were determined by fluorescence of Fluo-8. (G) 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.
Fig 10.
Model of PRRSV inducing autophagy via ER stress-induced calcium signaling pathway.
PRRSV attaches to and enters cells, followed by viral protein Nsp2 interacting with GRP78 and STIM1 and inducing ER stress. ER stress results in the STIM1 moving towards to the cell membrane, opening the SOCE channel, inducing Ca2+ influx, and finally activating the CaMKII-AMPK-mTOR-autophagy pathway.