Pseudorabies virus induces ferroptosis by disrupting iron homeostasis through activation of TfR1 and ferritinophagy

Abstract

Programmed cell death (PCD) refers to a regulated cellular process involving a cascade of biochemical reactions and molecular mechanisms, commonly including apoptosis, necroptosis, and pyroptosis. Ferroptosis is a recently identified form of PCD distinguished by its dependence on iron. Emerging evidence underscores the significance of ferroptosis in viral infections; however, its role in Pseudorabies virus (PRV) infection, an enveloped double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, remains poorly understood. Here, we demonstrate that PRV infection induces multiple forms of PCD, including ferroptosis, which is characterized by mitochondrial shrinkage, lipid peroxidation, ferrous iron (Fe²⁺) accumulation, and elevated levels of reactive oxygen species (ROS). Ferroptosis facilitates PRV replication, with iron overload playing a crucial role. Mechanistically, we show that transferrin receptor 1 (TfR1) and ferritinophagy are involved in PRV-induced iron overload. Specifically, PRV infection upregulates TfR1 expression via hypoxia-inducible factor-1β (HIF-1β) and promotes its translocation to the cell membrane through Rab11a, thereby enhancing the cellular import of extracellular ferric iron (Fe³⁺). In parallel, PRV activates ferritinophagy to degrade ferritin heavy chain 1 (FTH1) via selective autophagy receptors, nuclear receptor coactivator 4 (NCOA4) and Tax1-binding protein 1 (TAX1BP1), further contributing to intracellular iron accumulation. Altogether, these findings demonstrate that PRV induces ferroptosis by disrupting iron homeostasis through TfR1 activation and ferritinophagy induction, providing novel insights into the pathogenesis of PRV and other herpesviruses.IMPORTANCEFerroptosis is an iron-dependent form of non-apoptotic cell death that primarily involves iron overload, lipid peroxidation, and suppression of antioxidant systems. Increasing evidence indicates that ferroptosis plays an important role in viral infections. In this study, we show that PRV induces ferroptosis by disrupting iron homeostasis through TfR1 activation and ferritinophagy induction. On one hand, PRV infection upregulates TfR1 expression through HIF-1β and facilitates TfR1 translocation to the cell membrane via Rab11a, leading to enhanced import of extracellular Fe³⁺ into cells. On the other hand, PRV exploits the selective autophagy receptors NCOA4 and TAX1BP1, which strengthens the interaction between NCOA4, TAX1BP1, and FTH1, triggering ferritinophagy and increasing intracellular Fe²⁺ levels. Collectively, these findings enrich the understanding of the mechanism by which PRV induces ferroptosis, shedding new light on PRV and other alpha-herpesvirus infections.

Keywords: PRV; TfR1; ferritinophagy; ferroptosis; iron homeostasis; programmed cell death.

Fig 1

PRV induces ferroptosis. (A) N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h, and images were obtained by using a transmission electron microscope at 20,000 × magnification. Yellow arrows indicate normal mitochondria in mock-infected cells, and red arrows indicate mitochondrial atrophy in PRV-infected cells. Representative images are shown. Scale bar, 500 nm. (B) The diameters of mitochondria in N2a cells were quantified. (C) N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.01, 0.1, or 1) or mock infection and additional treatment of Fer-1 (80 µM) or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. N2a cells were mock- or PRV-infected (MOI = 0.01, 0.1, or 1) for 24 h or treated with RSL3 (10 µM) as positive control. (D) LDH released into the supernatants was determined by a cytotoxicity assay, with LDH in mock-infected cells defined as 1. (E) MDA concentrations in cell lysates were determined using MDA assay. (F) Lipid peroxidation levels in cells were measured using the fluorescent probe C11-BODIPY 581/591. (G) Ferrous iron concentrations in cell lysates were determined using ferrous iron colorimetric assay. (H) ROS levels in cells were determined using a ROS assay. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 2

Ferroptosis promotes PRV replication. (A) Cells were treated with different concentrations of Fer-1 (40 or 80 µM) or vehicle (DMSO) for 24 h. Cell viability of N2a cells was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. (B) N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 (80 µM) or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. (C) At 24 hpi, cells and supernatants were harvested, and virus titers were determined in Vero cells using a plaque assay. (D) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. (E) N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection or mock infection and additional treatment of Fer-1 or vehicle. At 6 or 12 hpi, apoptosis was assessed using Annexin V-FITC/PI staining and analyzed by fluorescence-activated cell sorting (FACS). The values in each panel represent the percentage of viable cells. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 3

PRV replication depends on extracellular iron concentration. N2a cells were treated with either 15 µM FAC, 50 µM DFOM, or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of FAC/DFOM or vehicle. At 24 hpi, the following assays were performed: (A, G) Cell viability was determined using a CCK-8 assay, with vehicle-treated cells defined as 100%. (B, H) MDA concentrations in cell lysates were determined using MDA assay. (C, I) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. (D, J) ROS levels in cells were determined using a ROS assay. (E, K) Cells and supernatants were harvested, and virus titers were determined in Vero cells using a plaque assay. (F, L) Cell lysates were analyzed by Western blot for gB and β-actin. The protein levels were quantified using ImageJ and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 4

PRV infection induces ferroptosis through disruption of iron metabolism. (A) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for TfR1, FTH1, VP16, and β-actin. TfR1-knockdown (B), FTH1-overexpression (H), and wild-type N2a cells were treated with Fer-1 (80 µM) or the vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. TfR1-knockdown, FTH1-overexpression and wild-type N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. MDA concentrations in TfR1-knockdown (C) and FTH1-overexpression (I) cell lysates were determined. Ferrous iron concentrations in TfR1-knockdown (D) and FTH1-overexpression (J) cell lysates were determined. ROS levels in TfR1-knockdown (E) and FTH1-overexpression (K) cells were determined. Cells and supernatants were harvested, and total virus titers in TfR1-knockdown (F) and FTH1-overexpression (L) cells were determined in Vero cells using a plaque assay. Cell lysates of TfR1-knockdown (G) and FTH1-overexpression (M) were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 5

HIF-1β promotes TfR1 mRNA expression upon PRV infection. (A) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. RNA was extracted for RT-PCR analysis of TfR1 and RNA18s rRNA, and cell lysates were analyzed by Western blot for TfR1 and β-actin. (B) N2a cells were mock- or PRV-infected (MOI = 1) at various times. Cell lysates were analyzed by western blot for HIF-1α, HIF-1β, IRP1, IRP2, TfR1, VP16, and β-actin. HIF-1α-knockdown (C), HIF-1β-knockdown (D), IRP1-knockdown (E), IRP2-knockdown (F), and Wild-type N2a cells were harvested for RNA extraction and RT-PCR analysis of TfR1 and RNA18s rRNA, and cell lysates were analyzed by western blot for TFR1 and β-actin. (G) HIF-1β-knockdown cells and wild-type N2a cells were treated with Fer-1 or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 (80 µM) or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. Wild-type and HIF-1β-knockdown N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. (H) MDA concentrations in cell lysates were determined using MDA assay. (I) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. (J) ROS levels were determined using a ROS assay. (K) Cells and supernatants were harvested, and total virus titers were determined in Vero cells using a plaque assay. (L) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 6

Rab11a promotes endosomal trafficking of TfR1 upon PRV infection. N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. (A) Cell lysates were analyzed by western blot for Rab11a, TfR1, VP16, and β-actin. (B) Cell lysates were co-immunoprecipitated (co-IP) with anti-TfR1 antibody and then subjected to western blot analysis for Rab11a, TfR1, VP16, and β-actin. The grayscale intensity in the mock-infected group was set at 1.00 in the co-IP analysis. (C) N2a cells were mock-infected and infected with PRV at an MOI = 1 for 6 h or 12 h. Cells were stained for TfR1, Rab11a, and DAPI and performed using the Duolink In Situ Detection Reagents Green. Then observed by confocal microscopy. Scale bar, 10 µm. (D) Rab11a-knockdown and wild-type N2a cells were mock- or PRV-GFP-infected (MOI = 1) for 12 h. Cells were stained for TfR1and DAPI and observed by confocal microscopy. Scale bar, 10 µm. (E) Rab11a-knockdown cells and wild-type N2a cells were treated with Fer-1 (80 µM) or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. Wild-type and Rab11a-knockdown N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. (F) MDA concentrations in cell lysates were determined using MDA assay. (G) Ferrous iron concentrations in cell lysates were determined using a ferrous iron colorimetric assay. (H) ROS levels were determined using a ROS assay. (I) Cells and supernatants were harvested, and total virus titers were determined in Vero cells using a plaque assay. (J) Cell lysates were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 7

PRV induces ferritinophagy. (A) N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for NCOA4, TAX1BP1, FTH1, VP16, and β-actin. (B) N2a cells were mock- or PRV-infected (MOI = 1) for 2 h and then treated with 5 mM 3-MA or the vehicle. At 12 hpi, cell lysates were analyzed by western blot for NCOA4, TAX1BP1, FTH1, LC3, VP16, and β-actin. (C) N2a cells were transfected with FLAG-FTH1 (1 µg) for 24 h, and then mock- or PRV-GFP-infected (MOI = 1). At 12 hpi, cells were stained for FTH1, LAMP1, and DAPI and observed by confocal microscopy. Scale bar, 10 µm. (D) N2a cells were mock- or PRV-infected (MOI = 1) for 2 h and then treated with 5 mM 3-MA or the vehicle. At 12 hpi, cell lysates were precipitated (co-IP) with anti-NCOA4 antibody and analyzed by western blot analysis for TAX1BP1, FTH1, NCOA4, VP16, and β-actin. The grayscale intensity was set at 1.00 for the mock-infected group. (E) NCOA4-knockdown and wild-type N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for NCOA4, FTH1, LC3, VP16, and β-actin. (F) TAX1BP1-knockdown and wild-type N2a cells were mock- or PRV-infected (MOI = 1) for 12 h. Cell lysates were analyzed by western blot for TAX1BP1, FTH1, LC3, VP16, and β-actin. NCOA4-knockdown, TAX1BP1-knockdown, and wild-type N2a cells were mock- or PRV-infected (MOI = 0.1) for 24 h. NCOA4-knockdown (G) and TAX1BP1-knockdown (M) cells and wild-type N2a cells were treated with Fer-1 (80 µM) or vehicle (DMSO) for 2 h, followed by PRV infection (MOI = 0.1) or mock infection and additional treatment of Fer-1 or vehicle. At 24 hpi, cell viability was determined by CCK-8 assay, with the level of cell viability in cells treated with vehicle defined as 100%. MDA concentrations in NCOA4-knockdown (H) and TAX1BP1-knockdown (N) cell lysates were determined using MDA assay. Ferrous iron concentrations in NCOA4-knockdown (I) and TAX1BP1-knockdown (O) cell lysates were determined using a ferrous iron colorimetric assay. ROS levels in NCOA4-knockdown (J) and TAX1BP1-knockdown (P) cells were determined using a ROS assay. Cells and supernatants were harvested, and total virus titers of NCOA4-knockdown (K) and TAX1BP1-knockdown (Q) cells were determined in Vero cells using a plaque assay. Cell lysates of NCOA4-knockdown (L) and TAX1BP1-knockdown (R) cells were analyzed by western blot for gB and β-actin. The protein levels were quantified by Image J and normalized to β-actin. The data shown are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experimental data are representative of results from three independent experiments.

Fig 8

Schematic of the signaling pathway through which PRV infection disrupts cellular iron homeostasis, ultimately leading to the occurrence of ferroptosis. Black arrows indicate that PRV infection promotes the translocation of TfR1 to the cell membrane, increasing the influx of extracellular Fe³⁺. Blue arrows indicate that PRV infection utilizes the selective autophagy receptors NCOA4 and TAX1BP1 to degrade FTH1, inducing the process of ferritinophagy. This model was created by the author, with parts of the figure derived from Servier Medical Art (https://smart.servier.com), licensed under CC BY 4.0.