The Hemagglutinin of Influenza A Virus Induces Ferroptosis to Facilitate Viral Replication

Abstract

Ferroptosis is a novel form of cell death caused by the accumulation of lipid peroxides in an iron-dependent manner. However, the precise mechanism underlying the exploitation of ferroptosis by influenza A viruses (IAV) remains unclear. The results demonstrate that IAV promotes its own replication through ferritinophagy by sensitizing cells to ferroptosis, with hemagglutinin identified as a key trigger in this process. Hemagglutinin interacts with autophagic receptors nuclear receptor coactivator 4 (NCOA4) and tax1-binding protein 1 (TAX1BP1), facilitating the formation of ferritin-NCOA4 condensates and inducing ferritinophagy. Further investigation shows that hemagglutinin-induced ferritinophagy causes cellular lipid peroxidation, inhibits aggregation of mitochondrial antiviral signaling protein (MAVS), and suppresses the type I interferon response, thereby contributing to viral replication. Collectively, a novel mechanism by which IAV hemagglutinin induces ferritinophagy resulting in cellular lipid peroxidation, consequently impairing MAVS-mediated antiviral immunity, is revealed.

Keywords: autophagy; ferroptosis; influenza a virus; innate immunity; mitochondrial antiviral signaling protein (MAVS).

Figure 1

IAV induces cellular ferroptosis in vitro. A549 cells were treated with RSL3 (2 µm) or infected with PR8 H1N1 virus (MOI = 0.1) for 24 hpi. A) Cell viability was determined using a CCK‐8 kit. B) The intracellular GSH levels were determined by GSH assay kit. C) The cellular concentration of MDA was used to measure the level of lipid peroxidation by MDA assay kit. D) The ferrous iron concentration in cell lysates was determined by iron assay kits. E) The cell breakage was taken with PR8 H1N1 virus (MOI = 0.1) infection or RSL3 (2 µm) treatment. Cell morphology was observed in BF, and dead cells were labeled with PI. Scale bar = 100 µm. F) The H2DCFDA probe‐labeled intracellular ROS levels were detected by flow cytometry, and mean fluorescence intensity (MFI) was also analyzed. G and H) The C11 BODIPY probe stained intracellular lipid ROS, which were subsequently detected by confocal microscopy as well as flow cytometry, respectively, and the MFI of C11 BODIPY was also analyzed. Scale bar = 10 µm. Data were shown as means ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (***p < 0.001).

Figure 2

Inhibition of ferroptosis restricts IAV replication. A) The cell viability of A549 cells was measured by CCK‐8 after treatment with different concentrations of Fer‐1 for 24 h. B–E) A549 cells were pretreated with Fer‐1 (5 µm) or vehicle (DMSO) for 2 hpi, followed by infected A549 cells with PR8 H1N1 virus (MOI = 0.1) in Fer‐1 (5 µm) or vehicle (DMSO) treatment. B) Cell viability was determined using a CCK‐8 kit. C) The cellular concentration of MDA was used to measure the level of lipid peroxidation by MDA assay kit. D) The ferrous iron concentration in cell lysates was determined by iron assay kits. E)The cell breakage was visualized by fluorescence microscopy, the cell morphology was shown in BF, and the dead cells were stained by PI. Scale bar = 100 µm. F–G) A549 cells were pretreated with Fer‐1 (5 µm) or vehicle (DMSO) for 2 hpi, followed by infected A549 cells with PR8 H1N1 virus (MOI = 0.1) in Fer‐1 (5 µm) or vehicle (DMSO) treatment, and cell supernatants and lysates were harvested at 12, 24, and 36 hpi, respectively. F) Viral titers in MDCK cell supernatants were determined by TCID₅₀. G) The viral protein NP expression detected by western blotting. Data were shown as means ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3

Ferritinophagy‐induced ferroptosis benefits IAV replication. A,B) The A549 cells were infected with PR8 H1N1 virus (MOI = 0.1) and cell lysates were harvested at 12, 24, and 36 hpi. A) The protein expression of ferroptosis genes was detected by western blotting with PR8 H1N1 infection of A549 cells. B) The protein expression of ferritinophagy genes was determined by western blotting with PR8 H1N1 virus infection of A549 cells. C) Confocal analysis of FTH1 expression levels in knockdown NCOA4 cells. Transfected with NC or Si NCOA4 in A549 cells, and infected with or without PR8 H1N1 virus (MOI = 0.1) 24 hpi following transfection, FTH1 (Green), lysosomes (Red), and cell nucleus (Blue) were stained. Scale bar = 20 µm. D) The protein expression of ferritinophagy genes was analyzed by western blotting for PR8 H1N1 virus infection treated with CQ. A549 cells infected with PR8 H1N1 virus (MOI = 0.1) at the treatment of CQ (100 µM) and cell lysates were harvested at 12, 24, and 36 hpi for western blotting analysis. E,F) PR8 H1N1 virus (MOI = 0.1) infected A549WT cells or NCOA4 KO cells, and cell supernatants as well as lysates were harvested. E) The protein expression of ferritinophagy genes and the viral nucleoprotein with PR8 H1N1 virus infection was analyzed by western blotting in NCOA4 KO cells. F) Viral titers in MDCK cell supernatants were determined by TCID₅₀. Data were shown as mean ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (**p < 0.01; ***p < 0.001).

Figure 4

Hemagglutinin induces ferritinophagy by interacting with TAX1BP1 and NCOA4. A) The degradation of ferritin by PR8 H1N1 viral proteins. HEK293T cells were transfected with nine plasmids encoding PR8 H1N1 viral proteins, and cell lysates were harvested at 24 h for western blotting to detect the protein expression of FTH1. *Target bands. B–F) HEK293T cells were transfected with PR8 HA, and cells or cell lysates were harvested at 24 h post‐transfection. B) Intracellular MDA levels were determined by MDA kit. C) Cellular GSH levels were determined by GSH assay kit. D) Intracellular ferrous iron concentration was determined by iron assay kit. E) Intracellular lipid ROS levels were determined with C11 BODIPY probe staining by flow cytometry. F) Intracellular free ferrous iron was determined with FerroOrange probe staining by flow cytometry. The MFI of C11 BODIPY and FerroOrange were analyzed. G) The protein expression of ferritinophagy genes following transfection with PR8 HA in case of autophagy inhibition HEK293T cells were transfected with PR8 HA for 6 h and then treated with or without CQ (100 µm) for 24 h, the cell lysates were harvested by western blotting analysis. H) An interaction network analysis of PR8 HA. I,J) The exogenous interactions of PR8 HA with NCOA4. HEK293T cells were transfected with Flag‐HA_PR8 and HA‐NCOA4, and cell lysates were harvested at 24 h for Co‐IP and western blotting analysis. K,L) The endogenous interactions of PR8 HA with NCOA4. K) The Flag‐NCOA4 was transfected in HEK293T cells, treated with CQ (100 µm) for 24 h at 6 h post‐transfection, cell lysates were used for Flag‐NCOA4 pull‐down and western blotting analysis. L) PR8 H1N1 virus (MOI = 0.1) was infected with HEK293T cells, and cell lysates used in PR8 HA pull‐down and western blotting analysis. M) Confocal analysis of the co‐localization of PR8 HA with NCOA4. HEK293T cells were transfected with vector or Flag‐HA_PR8 (Red), then indicated with the relevant antibodies for Flag‐HA_PR8 (Red) and NCOA4 (Green), the cell nucleus was stained with DAPI (Blue). The fluorescence intensity profiles of Flag‐HA_PR8 and NCOA4 were measured along the lines drawn by Image J. Scale bar = 5 µm. N) The effects of PR8 HA on Ferritin‐NCOA4 condensates. HEK293T cells were transfected with a vector or Flag‐HA_PR8 and GFP‐FTH1 (Green). NCOA4 (Red) and Flag‐HA_PR8 (Violet) were detected using specific antibodies, and the cell nuclei were stained with DAPI. Subsequently, 100 condensates with diameters larger than 1 µm were randomly statistically analyzed. Scale bar = 2 µm. O,P) The exogenous interaction of the PR8 HA with TAX1BP1. HEK293T cells were transfected with Flag‐HA_PR8 and HA‐TAX1BP1, followed by harvesting of cell lysates for Co‐IP and western blotting analysis. Q) Confocal analysis of co‐localization for PR8 HA with TAX1BP1. HEK293T cells were transfected with vector or Flag‐HA_PR8 and HA‐TAX1BP1, the relevant antibodies indicated Flag‐HA_PR8 (Red), HA‐TAX1BP1 (Green), and the cell nucleus was stained with DAPI. The fluorescence intensity profiles of Flag‐HA_PR8 and HA‐TAX1BP1 were measured along the lines drawn by Image J. Scale bar = 10 µm. R) The effect of PR8 HA with regard to the protein expression of TAX1BP1‐NCOA4. HEK293T cells were transfected with vector or HA‐NCOA4 and Flag‐TAX1BP1, and cell lysates were analyzed by western blotting with or without Flag‐HA_PR8 present. Data were shown as mean ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5

Lipid peroxidation caused by IAV‐induced ferritinophagy inhibits IFN‐β levels. A) qRT‐PCR analysis of IFNβ mRNA levels by PR8 H1N1 virus‐stimulated in NCOA4 KO cell lines. A549 WT or NCOA4 KO cells were infected with or without PR8 H1N1 virus (MOI = 0.1) for 24 hpi. qRT‐PCR analysis of IFNβ mRNA levels was performed. B) qRT‐PCR analysis of IFNβ mRNA levels in NCOA4 KO cells with Poly (I:C)‐stimulated. A549 WT or NCOA4 KO cells were stimulated with Poly (I:C) (200 ng) for 12 h. qRT‐PCR analysis of IFNβ mRNA levels was performed. C,D) IFNβ mRNA levels by PR8 H1N1 virus (MOI = 0.1) stimulated A549 cells with or without lipid peroxidation inhibitor treatment. C) A549 cells with or without DFO (100 µm) treatment, and the IFNβ mRNA levels were analyzed by qRT‐PCR. D) A549 cells with or without Fer‐1 (5 µm) treatment. the IFNβ mRNA levels were analyzed by qRT‐PCR. E,F) HEK293T cells were transfected with PR8 HA, followed by Poly (I:C) (200 ng) stimulation for 12 h, and IFNβ levels were analyzed by qRT‐PCR assay and luciferase assay, respectively. Data were shown as means ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant).

Figure 6

Lipid peroxidation resulting from IAV hemagglutinin impairs MAVS‐mediated antiviral immunity. A) The IFNβ promoter activity induced by MAVS in HEK293T WT cells or NCOA4 KO cells, with or without transfection of PR8 HA. HEK293 WT or KO cells were transfected with vector or PR8 HA and MAVS, followed by IFNβ promoter activity determined by luciferase assay. B–D) The effects of PR8 HA on MAVS‐induced IFNβ promoter activity with and without lipid peroxidation inhibition. HEK293T cells were transfected with either vector or PR8 HA and MAVS. After 6 h post‐transfection, the cells were treated with NCOA4‐9a (2.5 µm), DFO (100 µm), or Fer‐1 (5 µm) for 24 h. The IFNβ promoter activity was detected by luciferase assay. E) Western blotting analysis was performed to investigate the impact of PR8 HA on MAVS aggregates. HEK293T cells were co‐transfected with vector or HA‐HA_PR8 and Flag‐MAVS in HEK293T cells, and cell lysates were harvested at 24 h for SDS‐PAGE and SDD‐AGE, followed by western blotting analysis. F) Western blotting analysis of the effect of PR8 HA for MAVS aggregates in NCOA4 KO cells. In HEK293T WT or NCOA4 KO cells, vector or HA‐HA_PR8 and Flag‐MAVS were co‐transfected separately, and cell lysates were harvested at 24 h post‐transfection for SDD‐AGE vs SDS‐PAGE, followed by western blotting analysis. G‐I) The effects of PR8 HA on MAVS aggregates with and without lipid peroxidation inhibition. HEK293T cells were transfected with either vector or PR8 HA and MAVS. After 6 h of transfection, the cells were treated with NCOA4‐9a (2.5 µm), DFO (100 µm) or Fer‐1 (5 µm) for 24 h. Cell lysates were harvested at 24 h post‐transfection for SDD‐AGE vs SDS‐PAGE, followed by western blotting analysis. J) 4‐HNE levels were analyzed by western blotting in HEK293T cells transfected with 1, 2, and 4 µg of PR8 HA. K‐M) HEK293T cells were transfected with Flag‐MAVS, followed by treatment with 0, 5, 10 and 20 µm 4‐HNE for 12 h. Cell lysates were harvested for analysis. K) The IFNβ promoter activity induced by 4‐HNE on MAVS was determined by luciferase assay. L) The effects of 4‐HNE on MAVS aggregates were analyzed by western blotting. M) The ρ‐IRF3 level induced by 4‐HNE on MAVS was analyzed by western blotting. Data were shown as mean ± SEM (n = 3) from triplicate independent experiments, and significance was analyzed by two‐tailed Student's t‐test. (**p < 0.01; ***p < 0.001; ns, no significant).

Figure 7

IAV hemagglutinin‐induced lung ferroptosis impairs MAVS in vivo. A) The expression of Ad5‐HA_PR8 in A549 cells was analyzed by western blotting. The recombinant adenoviruses Ad5‐eGFP and Ad5‐HA_PR8 infected A549 cells (MOI = 0.1) and cell lysates were harvested at 48 hpi. Mock, Ad5‐eGFP served as a negative control. B‐G) Mice were infected with Ad5‐eGFP and Ad5‐HA_PR8, and ferroptosis was examined in mice lungs at 3 days (n = 5). B) Intracellular MDA levels were determined by MDA kit. C) Intracellular ferrous iron concentration was determined by iron assay kit. D) Intracellular lipid ROS levels were determined with C11 BODIPY probe staining by flow cytometry. E) Intracellular free ferrous iron was determined with FerroOrange probe staining by flow cytometry. The MFI of C11 BODIPY and FerroOrange were analyzed. F) Western blotting analysis of protein expression of ferritinophagy genes in mice lungs following Ad5‐HA_PR8 infection. G) Western blot analysis of the effects of MAVS aggregates in mice lungs after Adv‐HA_PR8 infection. Data were shown as mean ± SEM, and significance was analyzed by two‐tailed Student's t‐test. (**p < 0.01; ***p < 0.001).

Figure 8

A schematic representation of IAV hemagglutinin inhibiting MAVS‐mediated antiviral immunity by inducing ferritinophagy‐induced lipid peroxidation.