Fig 1.
PRRSV infection triggers mitochondrial fission.
(A-C) Marc-145 cells were infected with PRRSV (MOI = 0.1) for 24 h. (A) The cells were stained with MitoTracker Red, PRRSV-N antibody, and DAPI, followed by confocal microscopy. Enlarged images show the typical tubular mitochondrial network in uninfected cells and fragmented mitochondria in infected cells. Quantitative analysis of mitochondrial length is presented on the right. The lengths of mitochondria from 25 cells were quantified in each group. (B) The ultrastructure of mock-infected and PRRSV-infected cells was examined by electron microscopy. “N” represents nuclei. Zoomed-in images show normal elongated tubular mitochondria in uninfected cells and fragmented mitochondria with loss of cristae in infected cells. Scale bar = 2 μm. Quantification of mitochondrial length (n = 12) is shown on the right. (C) Mock-infected and PRRSV-infected cells were harvested for western blot analysis using antibodies against p-DRP1(S637), p-DRP1(S616), DRP1, PRRSV-N and β-actin. The levels of phosphorylated DRP1 were normalized to total DRP1 protein, while the levels of other proteins were normalized to β-actin. Data are expressed as means ± SD, n = 25 in A and n = 12 in B. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 2.
Mitochondrial fission facilitates PRRSV replication.
(A-F) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1) for 24 h with the indicated doses of Mdivi-1 (A-C) or siRNA (D-F). (A and D) Cell lysates were harvested for western blot analysis with antibodies against PRRSV-N, DRP1 and β-actin. The levels of proteins were normalized to β-actin. (B and E) Determination of the TCID50 of PRRSV in the cell supernatant. (C and F) Marc-145 cells were incubated with MitoTracker Red, PRRSV-N antibody and DAPI, followed by observation under a confocal microscope. Quantitative analysis of mitochondrial length is presented on the right. The lengths of mitochondria from 25 cells per group were quantified. Data are expressed as means ± SD, n = 3 in B and E and n = 25 in C and F. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 3.
Mitochondrial Ca2+ from IP3R channel mediates mitochondrial fission.
(A-D) Marc-145 cells were infected with PRRSV (MOI = 0.1) for 24 h with or without 2-APB treatment. (A) Ca2+ was detected by flow cytometry after Rhod-2 staining. (B) The cells were analyzed by confocal microscopy after staining with MitoTracker Red, PRRSV-N antibody and DAPI. The mitochondrial length was quantified from 25 cells per group (right panel). (C) The cell lysates were harvested for western blot analysis with antibodies against p-DRP1(S637), p-DRP1(S616), DRP1, PRRSV-N and β-actin. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. (D) Quantification of the TCID50 of PRRSV in cell supernatants. Data are expressed as means ± SD, n = 3 in A and D or n = 25 in B. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 4.
VDAC1-MCU channels mediate mitochondrial Ca2+ entry and mitochondrial fission.
(A-H) Marc-145 cells were infected with PRRSV (MOI = 0.1) for 24 h with or without VBIT-12 (A-D) or MCU-i4 (E-H) treatment. (A and E) Ca2+ was detected by flow cytometry after Rhod-2 staining. (B and F) The cells were analyzed by confocal microscopy after staining with MitoTracker Red, PRRSV-N antibody and DAPI. The mitochondrial length was quantified from 25 cells per group (right panel). (C and G) The cell lysates were harvested for western blot analysis with antibodies against p-DRP1(S637), p-DRP1(S616), DRP1, PRRSV-N and β-actin. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. (D and H) Quantification of the TCID50 of PRRSV in cell supernatants. Data are expressed as means ± SD, n = 3 in A, D, E and H or n = 25 in B and F. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 5.
PRRSV infection increases mitochondria-ER contacts mediated by SIGMAR1.
(A-C) Marc-145 cells were infected with PRRSV (MOI = 0.1) for 24 h. (A) The cell lysates were harvested for western blot analysis with antibodies against FUNDC1, SIGMAR1, PRRSV-N and β-actin. (B) Transmission electron microscopy was employed to observe the contact between the ER and mitochondria in mock-infected and PRRSV-infected cells. The mitochondrial-endoplasmic reticulum contacts were quantified using Image J (n = 20). (C) The cells were subjected to PLA with anti-Tom20 and anti-Calnexin antibodies. The number of PLA spots was quantified for 20 cells per group. (D-F) Marc-145 cells were transfected with control siRNA or siRNA targeting SIGMAR1 for 24 h prior to mock infection or infection with PRRSV (0.1 MOI) for an additional 24 hours. (D) Ca2+ levels were assessed using flow cytometry with Rhod-2 staining. (E) The mock-infected and PRRSV-infected cells were analyzed by confocal microscopy after staining with MitoTracker Red, PRRSV-N antibody and DAPI. The mitochondrial length was quantified from 25 cells per group (right panel). (F) The cell lysates were harvested for western blot analysis with antibodies against p-DRP1(S637), p-DRP1(S616), DRP1, PRRSV-N, SIGMAR1 and β-actin. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. Data are expressed as means ± SD, n = 20 in B and C, n = 3 in D or n = 25 in E. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 6.
The CaMKKβ-AMPK-DRP1 cascade mediates PRRSV-induced mitochondrial fission.
(A-D) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1), with or without Compound C (10 μM) or STO-609 (10 μM) treatment for 24 hours. (A and C) The cell lysates were collected and subjected to western blot analysis with antibodies against CaMKKβ (panel C only), p-AMPK, AMPK, p-DRP1 (S637), p-DRP1 (S616), DRP1, PRRSV-N and β-actin. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. (B and D) The cells were stained with MitoTracker Red, PRRSV-N antibody and DAPI, followed by confocal microscopy. The mitochondrial length was quantified from 25 cells per group (right panel). (E) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1) for 24 h, and then harvested for cytoplasmic and mitochondrial fractionation. Samples were subjected to western blot analysis with antibody against CaMKKβ, AMPK, DRP1, PRRSV-N, Tom20 and α-tubulin. The levels of proteins were normalized to Tom20 or α-tubulin. Data are expressed as means ± SD, n = 25 in B and D. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 7.
FIS1 and MiD49 as the adaptors of DRP1 mediates PRRSV-induced mitochondrial fission.
(A-B) Marc-145 cells were transfected with control siRNA or siRNA targeting FIS1 (A) or MiD49 (B) for 24 h, and then were either mock-infected or infected with PRRSV (0.1 MOI) for an additional 24 h. The cells were stained with MitoTracker Red, PRRSV-N antibody, and DAPI, followed by confocal microscopy. Quantitative analysis of mitochondrial length is presented for 25 cells per group (right panel). (C-D) Marc-145 cells were mock-infected or infected with PRRSV (MOI = 0.1) for 24 h. The cells were lysed and precipitated using anti-FIS1 or anti-MiD49 antibodies. The whole-cell lysates (WCL) and immunoprecipitated proteins were analyzed using specific antibodies. Anti-Rabbit immunoglobulin G (IgG) antibody was used as a negative control. (E and F) After 24 hours of siRNA FIS1 (E) and MiD49 (F) transfection, the cells were either mock-infected or infected with PRRSV (0.1 MOI) for an additional 24 hours, and then harvested for cytoplasmic and mitochondrial fractionation. Then samples were subjected to western blot analysis. The levels of proteins were normalized to Tom20 or α-tubulin. Data are expressed as means ± SD, n = 25 in A and B. *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 8.
Mitochondrial Ca2+ triggers PRRSV-induced mitophagy via the PINK1-Parkin pathway.
(A-D) Marc-145 cells were mock-infected or PRRSV-infected (MOI = 0.1) for 24 h with VBIT-12 (10 μM) or MCU-i4 (10 μM). (A) SOX was detected by flow cytometry of MitoSOX Red-stained cells. Statistical analysis of the positive cell population is presented (right panel). (B) The mitochondrial membrane potential was detected by flow cytometry of JC-1-stained cells. Statistical analysis of the positive cell population is presented (right panel). (C-D) Cell lysates were harvested for western blot analysis with antibodies against PINK1, Parkin, LC3, PRRSV-N and β-actin. (E-H) After 24 hours of siRNA transfection, the cells were either mock-infected or infected with PRRSV (0.1 MOI) for an additional 24 hours. (E-F) The cell lysates were harvested for western blot analysis with antibodies against PRRSV-N, PINK1 (for E), Parkin (for F) and β-actin. (G-H) Determination of the TCID50 of PRRSV in cell supernatants. Data are expressed as means ± SD, n = 3 in A, B, G and H. *p<0.05; **p < 0.01; ***p < 0.001. The levels of proteins were normalized to β-actin. The data are representative of results from three independent experiments.
Fig 9.
Mitochondrial Ca2+, mitochondrial fission and mitophagy promote glycolysis.
(A-F) Marc-145 cells were mock-infected or PRRSV-infected (MOI = 0.1) for 24 h without or with the addition of VBIT-12 (10 μM) or MCU-i4 (10 μM). OCR and ECAR assays were conducted, and statistical analysis was performed to assess cellular ATP production, basal respiration, basal glycolysis and compensatory glycolytic capacity. (G-R) After 24 hours of siRNA transfection, cells were either infected with PRRSV (0.1 MOI) or left uninfected for an additional 24 hours. OCR and ECAR measurements were conducted, and statistical analysis of the cellular ATP production capacity and basal respiration capacity or glycolytic capacity was conducted. Data are expressed as means ± SD (n = 3). *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 10.
PRRSV exploits glycolysis to promote its own replication.
(A-F) In Marc-145 cells, after drug treatment (VBIT-12, MCU-i4 or Mdivi-1) or siRNA transfection for 24 hours, followed by simulated or PRRSV infection for an additional 24 hours, the levels of HK or LDH were measured. (G) Marc-145 cells were infected with PRRSV (MOI = 0.1) for 24 hours, and then the intracellular lactate levels were measured. (H and I) Marc-145 cells were treated with lactate (20 mM) and then either mock-infected or infected with PRRSV (MOI = 0.1) for 24 hours. (H) The cell lysates were harvested for western blot analysis with antibodies against PRRSV-N and β-actin. The level of PRRSV-N was normalized to β-actin. (I) Determination of the TCID50 of PRRSV in the cell supernatant. Data are expressed as means ± SD (n = 3). *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 11.
PRRSV infection triggers mitochondrial fission and mitophagy mediated by mitochondrial Ca2+ in primary cells.
(A-C) PAMs were mock-infected or PRRSV-infected (MOI = 0.1) for 18 h. (A) The cells were stained with MitoTracker Red, PRRSV-N antibody and DAPI, followed by confocal microscopy. Quantitative analysis of mitochondrial length is presented on the right. The lengths of mitochondria from 20 cells were quantified for each group. (B) Cell lysates were harvested for western blot analysis. (C) Detection of mitochondrial Ca2+ using Rhod-2 staining by flow cytometry. (D and E) PAMs were exposed to PRRSV (MOI = 0.1) for 18 hours, either with or without Mdivi-1 (2.5 μM) treatment. (D) Western blot analysis was performed on the cell lysates by using antibodies against DRP1, PRRSV-N and β-actin. (E) Assessment of the TCID50 of PRRSV in the cell supernatant. (F and G) PAMs were either treated or not treated with VBIT-12 (5 μM), then mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. (F) Ca2+ levels were assessed using flow cytometry with Rhod-2 staining. (G) The cell lysates underwent western blot analysis using antibodies specific to DRP1 Ser637, DRP1 Ser616, DRP1, PRRSV-N and β-actin. (H) PAMs were mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. Collect cell lysates and perform western blot analysis using antibodies against PINK1, Parkin, LC3, PRRSV-N, and β-actin. (I-L) PAMs cells were transfected with siRNA targeting PINK1 (I and J) or Parkin (K and L) for 24 hours, followed by 18 hours of mock or PRRSV infection (MOI = 0.1). (I and K) Cell lysates were harvested for western blot analysis with antibodies against PINK1 or Parkin, PRRSV-N and β-actin. (J and L) Determination of the TCID50 of PRRSV in the cell supernatant. (M and N) PAMs were either treated or not treated with VBIT-12 (5 μM), then mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. (M) Cell lysates were harvested for western blot analysis with antibodies against PINK1, Parkin, LC3, PRRSV-N, and β-actin. (N) Determination of the TCID50 of PRRSV in the cell supernatant. Data are expressed as means ± SD, n = 20 in A or n = 3 in C, E, F, J, L, N. *p<0.05; **p < 0.01; ***p < 0.001. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. The data are representative of results from three independent experiments.
Fig 12.
PRRSV promotes its replication in primary cells by stimulating glycolysis.
(A-F) After treating PAMs with VBIT-12, cells were either mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. Subsequently, OCR and ECAR measurements were performed to assess cellular ATP production, basal respiration, basal glycolysis, and compensatory glycolysis capacity. (G and H) PAMs were pretreated with VBIT-12 (5 μM), followed by mock or PRRSV infection (MOI = 0.1) for 18 hours. Then, the levels of HK (G) and LDH (H) in the cells were measured. (I) After infecting PAMs with PRRSV at an MOI of 0.1 for 18 hours, lactate levels in the cells were measured using the CheKine Micro Lactate Assay Kit. (J and K) PAMs were pretreated with 10 mM lactate for 2 hours, followed by mock or PRRSV infection (MOI = 0.1) for 18 hours. (J) Cell lysates were collected for western blot analysis with antibodies against PRRSV-N and β-actin. The level of PRRSV-N was normalized to β-actin. (K) Quantification of the TCID50 of PRRSV in cell supernatants. Data are expressed as means ± SD (n = 3). *p<0.05; **p < 0.01; ***p < 0.001. The data are representative of results from three independent experiments.
Fig 13.
Model of calcium-mediated mitochondrial fission, mitophagy and glycolysis to facilitate PRRSV replication.
Upon cellular invasion, PRRSV induces ER stress and promotes the formation of MAMs via SIGMAR1, facilitating Ca2+ influx into the mitochondria through the IP3R-VDAC1-MCU channel. Excessive Ca2+ induces DRP1 S637 dephosphorylation and S616 phosphorylation via CaMKKβ-AMPK, leading to sustained mitochondrial fission, mitochondrial dysfunction, and activation of the PINK1-Parkin pathway for mitophagy clearance of damaged mitochondria. Concurrently, mitochondrial damage and mitophagy impair oxidative phosphorylation and enhance glycolysis, ultimately promoting PRRSV replication. Fig 13 was modified from Servier Medical Art (https://smart.servier.com/), licensed under a Creative Commons Attribution 4.0 International (CC BY) license (https://creativecommons.org/licenses/by/4.0/).