The glycoprotein 5 of porcine reproductive and respiratory syndrome virus stimulates mitochondrial ROS to facilitate viral replication

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant economic concern for the global swine industry due to its connection to serious production losses and increased mortality rates. There is currently no specific treatment for PRRSV. Previously, we had uncovered that PRRSV-activated lipophagy to facilitate viral replication. However, the precise mechanism that PRRSV used to trigger autophagy remained unclear. Here, we found that PRRSV GP5 enhanced mitochondrial Ca²⁺ uptake from ER by promoting ER-mitochondria contact, resulting in mROS release. Elevated mROS induced autophagy, which alleviated NLRP3 inflammasome activation for optimal viral replication. Our study shed light on a novel mechanism revealing how PRRSV exploits mROS to facilitate viral replication.

Keywords: ER-mitochondria contacts; IP3R; NLRP3 inflammasome; PRRSV; VDAC1; autophagy.

Fig 1

PRRSV infection induces the morphological alterations of mitochondria and ER, and ER-mitochondria contacts. (A) MARC-145 cells were mock-infected or infected with PRRSV (MOI = 1) for 24 h. The morphologies of Golgi (GM130), lysosomes (LAMP1), mitochondria (Tom20), and ER (calnexin) were monitored by immunofluorescence analysis. Scale bar: 10 µM. (B) MARC-145 cells were infected with PRRSV (MOI = 1) for 0–48 h. The morphology of mitochondria (Tom20) was monitored by immunofluorescence analysis. Scale bar: 10 µM. (C) Quantification of the fragmented, tubular, elongated, and aggregated mitochondria from (B) (n = 30). ^*P < 0.05, ^**P < 0.01. (D) MARC-145 cells were infected with PRRSV (MOI = 1) for 0–48 h. The morphology of ER (calnexin) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (E) Quantification of the percentage of ER tubule from (D) (n = 30). ^**P < 0.01, ^***P < 0.001. (F) MARC-145 cells were infected with PRRSV (MOI = 1) for 0–48 h. The co-localization of ER (calnexin) with mitochondria (Tom20) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (G) Quantification of the co-localization of ER with mitochondria from (F) (n = 30). ^**P < 0.01, ^***P < 0.001.

Fig 2

PRRSV GP5 is responsible for ER-mitochondria contact. (A) MARC-145 cells were transfected with EGFP, GP5-EGFP, or NSP2-EGFP plasmids for 24 h. The morphology of mitochondria (Tom20) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (B) Quantification of mitochondrial networks number from (A) (n = 30). ^***P < 0.001. (C) Quantification of the fragmented, tubular, elongated, and aggregated mitochondria from (A) (n = 30). ^***P < 0.001. (D) MARC-145 cells were transfected with EGFP, GP5-EGFP, or NSP2-EGFP plasmids for 24 h. The morphology of ER (calnexin) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (E) Quantification of the percentage of ER tubules from (D) (n = 30). ^***P < 0.001. ns, no significance. (F) MARC-145 cells were co-transfected with pDsRed2-ER and vector, Myc-GP5, or NSP2-HA for 24 h. The co-localization of ER (DsRed) with mitochondria (Tom20) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (G) Quantification of the co-localization of ER with mitochondria from (F) (n = 30). ^***P < 0.001. ns, no significance.

Fig 3

PRRSV GP5 alters mitochondrial dynamics. (A) MARC-145 cells were transfected with GP5-EGFP (0–4 µg) for 24 h. The mRNA levels of MFN1 were analyzed by qRT-PCR analysis. ^**P < 0.01, ^***P < 0.001. (B) MARC-145 cells were transfected with GP5-EGFP (0–4 µg) for 24 h. The mRNA levels of DRP1, MFF, and ATF4 were analyzed by qRT-PCR analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (C) MARC-145 cells were transfected with GP5-EGFP (0–4 µg) for 24 h. The mRNA levels of PARKIN and PINK1 were analyzed by qRT-PCR analysis. ^**P < 0.01, ^***P < 0.001. (D) MARC-145 cells were transfected with GP5-EGFP (0–8 µg) for 24 h. DRP1, p-DRP1, Tom20, and GP5-EGFP were analyzed by immunoblotting analysis. (E) MARC-145 cells were transfected with Myc-GP5 (0–4 µg) for 24 h. The mitochondrial membrane potential was analyzed by JC-1 staining. Scale bar: 10 µm. (F) Quantification of the ratio of aggregated and monomer JC-1 from (E). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (G) Cellular ATP was measured in MARC-145 cells transfected with GP5-EGFP (0–4 µg) for 24 h. ^**P < 0.01, ^***P < 0.001. (H) NAD⁺/NADH ratio was measured in MARC-145 cells transfected with GP5-EGFP (0–4 µg) for 24 h. ^**P < 0.01, ^***P < 0.001. (I) MARC-145 cells were transfected with Myc-GP5 (0–4 µg) for 48 h. Apoptosis assay was performed by Annexin V FITC and PI staining. ^*P < 0.05.

Fig 4

PRRSV GP5 interacts with IP3R and VDAC1 to induce Ca2⁺ efflux from ER into mitochondria. (A) MARC-145 cells were transfected with vector or Myc-GP5 for 24 h and then treated with CCCP (10 µM) to release mitochondrial Ca²⁺. (B) The shControl, shGP5, and shIP3R MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h and then treated with CCCP (10 µM) to release mitochondrial Ca²⁺. (C) shControl, shGP5, and shIP3R MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h and then treated with Tg (10 µM) to release ER Ca²⁺. (D) MARC-145 cells were mock-infected or infected with PRRSV (MOI = 1) for 24 h. The interaction of GP5 with IP3R was analyzed by Co-IP analysis. (E) The interaction of GP5 with IP3R was analyzed by Co-IP analysis in samples from mock-infected or PRRSV-infected porcine lungs. (F) MARC-145 cells transfected with EGFP-C1 and GP5-EGFP were assessed by immunostaining with antibody against IP3R (red). Scale bar, 10 µm. (G) Quantification of the co-localization of GP5-EGFP with IP3R from (F). ^***P < 0.001. (H) MARC-145 cells were transfected with FLAG-VDAC1 for 24 h and then mock-infected or infected with PRRSV (MOI = 1) for 24 h. The co-localization of FLAG-VDAC1, GP5, and mitochondria (Tom20) was monitored by immunofluorescence analysis. Scale bar: 10 µm. (I) MARC-145 cells were co-transfected with FLAG-VDAC1 and EGFP or GP5-EGFP plasmids for 24 h. The co-localization of FLAG-VDAC1, GP5-EGFP, and Tom20 was monitored by immunofluorescence analysis. Scale bar: 10 µm. (J) MARC-145 cells were transfected with GP5-EGFP (0–8 µg) for 24 h. The VDAC1 oligomers and GP5-EGFP were analyzed by immunoblotting analysis. (K) The VDAC1 oligomers were analyzed by immunoblotting analysis in samples from mock-infected or PRRSV-infected porcine lungs. (L) MARC-145 cells were mock-infected or infected with PRRSV (MOI = 1) for 24 h. The interaction of GP5 with VDAC1 was analyzed by Co-IP analysis. (M) The interaction of GP5 with VDAC1 was analyzed by Co-IP analysis in samples from mock-infected or PRRSV-infected porcine lungs. (N) shControl and shVDAC1 MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h and then treated with CCCP (10 µM) to release mitochondrial Ca²⁺. (O) shControl and shVDAC1 MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h and then treated with Tg (10 µM) to release ER Ca²⁺. (P) shControl and shGP5 MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h. The interaction of VDAC1 and IP3R was analyzed by PLA assay (left). Quantification of the relative number of VDAC1/IP3R dots per cell is shown on the right. Scale bar: 10 µm. ^***P < 0.001. (Q) MARC-145 cells were transfected with vector or GP5-EGFP plasmid for 24 h. The interaction of VDAC1 and IP3R was analyzed by PLA assay (left). Quantification of the relative number of VDAC1/IP3R dots per cell is shown on the right. Scale bar: 10 µm. ^***P < 0.001.

Fig 5

PRRSV GP5 induces ROS and activates autophagy through the AMPK/mTOR/ULK1 axis. (A) MARC-145 cells were transfected with GP5-EGFP (0–4 µg) or NSP2-HA (0–4 µg) for 24 h. Intracellular ROS was determined by DCFH-DA staining. Scale bar: 10 µm. (B) Quantification of the relative ROS levels from (A). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. ns, no significance. (C) shControl and shGP5 MARC-145 cells were infected with PRRSV (MOI = 1) for 24 h. Intracellular ROS was determined by DCFH-DA staining. Scale bar: 10 µm. (D) Quantification of the relative ROS levels from (C). ^***P < 0.001. (E) MARC-145 cells were transfected with GP5-EGFP (4 µg) and simultaneously treated with vehicle or NAC (10 mM) for 24 h. The subcellular localization of GP5-EGFP and NRF2 was detected by immunofluorescence analysis. Scale bar: 10 µm. (F) Quantification of cells with nuclear-localized NRF2 from (E). ^***P < 0.001. (G) MARC-145 cells were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with vehicle or NAC (10 mM) for 24 h. The mRNA levels of SOD-2 and HO-1 were analyzed by qRT-PCR analysis. ^*P < 0.05, ^***P < 0.001. (H) MARC-145 cells were infected with PRRSV (MOI = 1) and simultaneously treated with NAC (0–10 mM) for 48 h. Viral titers were assessed by TCID₅₀ assay. ^**P < 0.01, ^***P < 0.001. (I) MARC-145 cells were infected with PRRSV (MOI = 0–1) and treated with NAC (10 mM) as indicated for 48 h. LC3-I, LC3-II, P62, PRRSV-N, and GAPDH were analyzed by immunoblotting analysis. (J) MARC-145 cells were co-transfected with Myc-GP5 and LC3-GFP and simultaneously treated with NAC (0–10 mM) as indicated for 24 h. LC3 puncta was detected by fluorescent microscopy (left). Quantification of LC3 puncta per cell is shown on the right. Scale bar: 10 µm. ^***P < 0.001. ns, no significance. (K) MARC-145 cells were transfected with GP5-EGFP (0–8 µg) for 24 h. p-AMPK, AMPK, p-mTOR, mTOR, p-ULK1 S757, p-ULK1 S555, ULK1, GP5-EGFP, and GAPDH were analyzed by immunoblotting analysis. (L) MARC-145 cells were transfected with GP5-EGFP (8 µg) and treated with NAC (10 mM) for 24 h. p-AMPK, AMPK, p-mTOR, mTOR, p-ULK1 S757, p-ULK1 S555, ULK1, LC3-I, LC3-II, P62, GP5-EGFP, and GAPDH were analyzed by immunoblotting analysis. (M) MARC-145 cells were transfected with GP5-EGFP (4 µg) and treated with compound C (10 mM) for 24 h. p-AMPK, AMPK, p-mTOR, mTOR, p-ULK1 S757, p-ULK1 S555, ULK1, LC3-I, LC3-II, P62, GP5-EGFP, and GAPDH were analyzed by immunoblotting analysis.

Fig 6

PRRSV GP5-induced ROS activates NLRP3 inflammasome. (A, B) iPAMs were infected with PRRSV (MOI = 1) simultaneously treated with NAC (10 mM) as indicated for 0–48 h. IL-1β (A) and IL-18 (B) in the medium were measured by ELISA. ^***P < 0.001. (C, D) iPAMs were infected with PRRSV (MOI = 1) and simultaneously treated with NAC (10 mM) as indicated for 0–48 h. The mRNA levels of IL-1β (C) and IL-18 (D) were analyzed by qRT-PCR analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (E) iPAMs were infected with PRRSV (MOI = 1) and simultaneously treated with NAC (10 mM) as indicated for 48 h. IL-1β, pro-IL-1β, pro-caspase-1, caspase-1 P20, and GAPDH were analyzed by immunoblotting analysis. (F) Caspase-1 activity was assessed in iPAMs infected with PRRSV (MOI = 1) and simultaneously treated with NAC (10 mM) or YVAD-CHO (5 µM) as indicated for 24 h. ^***P < 0.001. ns, no significance. (G) The LDH activities were measured in the medium from iPAMs infected with PRRSV (MOI = 1) and simultaneously treated with NAC (10 mM) as indicated for 0–48 h. ^**P < 0.01, ^***P < 0.001. (H, I) iPAMs were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with NAC (10 mM) as indicated for 24 h. IL-1β (H) and IL-18 (I) in the medium were measured by ELISA. ^**P < 0.01, ^***P < 0.001. (J, K) iPAMs were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with NAC (10 mM) as indicated for 24 h. The mRNA levels of IL-1β (J) and IL-18 (K) were analyzed by qRT-PCR analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (L) iPAMs were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with NAC (10 mM) as indicated for 24 h. IL-1β, pro-IL-1β, pro-caspase-1, caspase-1 P20, and GAPDH were analyzed by immunoblotting analysis. (M) Caspase-1 activity was assessed in iPAMs cells transfected with GP5-EGFP (4 µg) and simultaneously treated with NAC (10 mM) or YVAD-CHO (5 µM) as indicated for 24 h. ^***P < 0.001. ns, no significance. (N) The LDH activities were measured in the medium from iPAMs transfected with GP5-EGFP (0–4 µg) and simultaneously treated with NAC (10 mM) as indicated for 24 h. ^**P < 0.01, ^***P < 0.001.

Fig 7

PRRSV GP5-induced autophagy antagonizes NLRP3 inflammasome. (A, B) The iPAMs were infected with PRRSV (MOI = 1) and simultaneously treated with 3MA (10 µM) as indicated for 0–48 h. The mRNA levels of IL-1β (A) and IL-18 (B) were analyzed by qRT-PCR analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (C, D) iPAMs were infected with PRRSV (MOI = 1) simultaneously treated with 3MA (10 µM) as indicated for 0–48 h and IL-1β (C) and IL-18 (D) in the medium were measured by ELISA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (E) iPAMs were infected with PRRSV (MOI = 1) and simultaneously treated with 3MA (10 µM) as indicated for 48 h. IL-1β, pro-IL-1β, pro-caspase-1, caspase-1 P20, and GAPDH were analyzed by immunoblotting analysis. (F, G) iPAMs were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with 3MA (10 µM) as indicated for 24 h. IL-1β (F) and IL-18 (G) in the medium were measured by ELISA. ^*P < 0.05, ^***P < 0.001. (H, I) iPAMs were transfected with GP5-EGFP (0–4 µg) and simultaneously treated with 3MA (10 µM) as indicated for 24 h. The mRNA levels of IL-1β (H) and IL-18 (I) were analyzed by qRT-PCR analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (J) iPAMs were transfected with GP5-EGFP (4 µg) and simultaneously treated with 3MA (10 µM) as indicated for 24 h. IL-1β, pro-IL-1β, pro-caspase-1, caspase-1 P20, and GAPDH were analyzed by immunoblotting analysis. (K) Caspase-1 activity was assessed in iPAMs transfected with GP5-EGFP (4 µg) and simultaneously treated with 3MA (10 µM) as indicated for 24 h. ^*P < 0.05, ^***P < 0.001. (L) iPAMs were infected with PRRSV (MOI = 1) and simultaneously treated with 3MA (10 µM) as indicated for 0–48 h. Viral titers were assessed by TCID₅₀ assay. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig 8

A schematic model showing PRRSV GP5 stimulates mROS to facilitate viral replication.