Antiviral effects of ferric ammonium citrate

Abstract

Iron is an essential nutrient for cell survival and is crucial for DNA replication, mitochondrial function and erythropoiesis. However, the immunological role of iron in viral infections has not been well defined. Here we found the iron salt ferric ammonium citrate (FAC) inhibited Influenza A virus, HIV virus, Zika virus, and Enterovirus 71 (EV71) infections. Of note, both iron ion and citrate ion were required for the antiviral capability of FAC, as other iron salts and citrates did not exhibit viral inhibition. Mechanistically, FAC inhibited viral infection through inducing viral fusion and blocking endosomal viral release. These were further evidenced by the fact that FAC induced liposome aggregation and intracellular vesicle fusion, which was associated with a unique iron-dependent cell death. Our results demonstrate a novel antiviral function of FAC and suggest a therapeutic potential for iron in the control of viral infections.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

FAC inhibited influenza A virus infection in vitro and in vivo. a A549 cells were infected with PR8 (MOI = 0.1) ± (with or without) FAC (100 μM). 24 h later, supernatants were subject to viral titer assay. b A549 cells were infected with PR8 (MOI = 0.1), treated with increasing doses of FAC (μM). 12 h later, viral RNA loading was analyzed through real-time PCR. c, d 6–8-week old female C57BL/6 mice were intranasally infected with 1 × LD50 influenza A virus PR8 diluted in 25 μl PBS or PBS with FAC (15 mM) (each group, n = 9). Mice weight change (c) and survival rate (d) were recorded. Mice with relative weight lower than 75% were euthanatized and considered as death. P < 0.0001 in (c) (PBS versus FAC, 2-way ANOVA test). P = 0.0033 in (d) (PBS versus FAC, log-rank (Mantel-Cox) test). e–h Mice were infected as in (c) 25 μl PBS or PBS with FAC (15 mM) was intranasally delivered at day 2 post infection. Whole lung and bronchoalveolar lavage fluids (BALFs) of mice were collected at days 3 and 6. The viral RNA loading in lung was analyzed by real-time PCR (e). IL-1β, IL-6, TNF-α, and CCL2 in the whole lung and BALF were analyzed via ELISA (f). Infiltrated total cells and neutrophils (Ly-6G+) in BALFs were analyzed through flow cytometry (g). Lung tissue inflammation was visualized via H&E staining (left) (h), arrows indicate tissue damage. Ctrl, mice without infection or treatment; Scale bars, 200 μm. Histological scores (right) were made by estimating lung inflammation levels by a pathologist blinded to the study (0 = absent; 1 = light; 2 = moderate; 3 = severe). Data are representative of at least two independent experiments in (a, b). Error bars represent SEM in (c) or SD in other panels

Fig. 2

FAC inhibited HIV, ZIKV and EV71 infections.a Human PBMC-differentiated dendritic cells were infected with HIV-1 (Gag/p24 100 ng/ml) ± FAC (100 μM). 8, 24, 48, and 72 h later, HIV Gag RNA loading was analyzed through real-time PCR. b Vero cells were infected with ZIKV (MOI = 0.01) ± FAC (100 μM). Seventy two-hour later, viral titers in supernatants were analyzed. c U251 cells were infected with Zika virus (MOI = 0.1) ± FAC (100 μM). 24, 48 and 72 h later, viral RNA loading was analyzed through real-time PCR. d–f RD cells were infected with EV71 (MOI = 0.1) ± FAC (100 μM) for 8 h. Viral RNA loading was detected via real-time PCR (d). Cytopathy images were captured through bright field microscopy (e). Scale bars, 100 μm. Viral VP1 protein expression was detected via western blot (f). g RD cells were infected with EV71 (MOI = 0.1) and treated with increasing doses of FAC (μM). 1 h later, viruses were washed out and FAC was supplemented. 9 h later, viral titers in supernatant were analyzed through TCID50 method. h, i 6–8-week old female IFNRα/β−/−IFNγ−/− mice were intraperitoneally infected with 40 PFU ZIKV diluted in 800 μl PBS or PBS with FAC (15 mM) (each group, n = 5). Mice weight change (h) and survival rate (i) were recorded. P < 0.0001 in (h) (PBS versus FAC, 2-way ANOVA test). P < 0.05 in (i) (PBS versus FAC, log-rank (Mantel-Cox) test). Data are representative of at least two independent experiments in (a–g). Error bars represent SEM in (h) or SD in other panels

Fig. 3

Specific iron salt inhibited viral infection.a PR8 (MOI = 0.1)-infected A549 cells were treated with FAC (100 μM) and deferoxamine (DFO, 100 μM). 10 h later, viral RNA was analyzed through real-time PCR. b A549 cells were infected with PR8 and treated with indicated compounds (50 μM) or compounds combinations (50 μM + 50 μM). 12 h later, viral RNA was detected via real-time PCR. c, d RD cells were infected with EV71 and treated with indicated compounds (100 μM) or compounds combinations (100 μM + 100 μM). 12 h later, viral RNA was detected via real-time PCR (c); cytopathy images were captured through bright field microscopy (d). Scale bars, 100 μm. e A549 cells were infected as in (b) and treated with indicated compounds combinations (μM). Viral RNA was detected via real-time PCR. f RD cells were infected with EV71 and treated with indicated compounds combinations (μM). Twelve-hour later, viral RNA was detected via real-time PCR. Data are representative of three independent experiments. Error bars represent SD

Fig. 4

FAC-induced fusion of influenza A virus or liposome.a PR8-infected A549 cells were treated with FAC at indicated time points post infection. Viruses were washed out at 3 h post infection and cells were analyzed for viral RNA at 12 h post infection through real-time PCR. b A549 cells and PR8 were incubated at 4 °C ± FAC for 1 h and cells were washed and analyzed for viral RNA through real-time PCR. c PR8-infected A549 cells were treated with or without FAC for 1 h. Cells were analyzed under confocal microscopy of PR8 NP protein. Scale bars, 20 μm. d PR8-infected A549 cells were treated in the presence or absence of FAC for 1 h. Cells were analyzed via electron microscopy. Arrows indicate endocytosing and endocytosed viruses in the PR8 panels (left, upper and lower images, scale bars: 0.2 μm). In the PR8 + FAC panels, upper image shows fusion viruses (right, upper panel, scale bar: 0.2 μm); lower image shows fusion viruses on the cell surface (right, lower panel, scale bar: 1 μm). e A549 cells and PR8 virus were incubated at 37 °C ± FAC for 3 h. Cells were washed and analyzed for viral RNA through real-time PCR. f PR8 in solution with or without FAC was incubated for 1 h at room temperature, centrifuged, and analyzed by negative-stain electron microscopy. g Liposome ± FAC were incubated for 1 h at 37 °C, stained with DiD (50 μM), and analyzed through bright field microscopy. Scale bars, 20 μm. h Hela cells were transfected by liposome and poly(dA:dT)-rhodamine mix ± FAC for 6 h. Cells were analyzed via confocal microscopy. Scale bars, 10 μm. Data are representative of three independent experiments. Error bars represent SD

Fig. 5

FAC-induced intracellular vesicle fusion.a PR8-infected A549 cells were treated in the presence or absence of FAC for 6 h or 12 h. Cells were analyzed under confocal microscopy for PR8 NP protein. Scale bars, 20 μm. b PR8-infected Hela cells were treated in the presence or absence of FAC for 6 h. Cells were analyzed under confocal microscopy for PR8 NP protein and RAB7. Scale bars, 10 μm. c A549 cells were infected with PR8 ± FAC for 6 h and analyzed via electron microscopy. Endosomes (irregular circles, white inside) and endolysosomes (irregular circles, dark inside) were marked by white lines. N nucleus. M mitochondrion. d Hela cells were treated with or without FAC for 6 h and analyzed through confocal microscopy for RAB7. Average RAB7-positive vesicle area and total RAB7 positive vesicle area per cell were statistically analyzed. Scale bars, 20 μm. e Hela cells were treated with Dynasore (Dyn) and/or FAC for indicated time. Cells were stained with 0.1% crystal violet and analyzed under bright field microscopy. Scale bars, 20 μm. Data are representative of three independent experiments. Error bars represent SD