Respiratory Syncytial Virus-Induced Oxidative Stress Leads to an Increase in Labile Zinc Pools in Lung Epithelial Cells

FIG 1. RSV infection leads to increases in labile zinc in A549 cells. A549 cells were infected with UV-inactivated RSV (UV-RSV) or wild-type RSV at an MOI of 0.3, with IAV at an MOI of 1, or with DENV at an MOI of 5, as indicated. Labile zinc levels were measured at 24 h p.i. and 48 h p.i. using FLZ-3 (A) and ZP-1 (B) by flow cytometry. A549 cells were infected with RSV at MOIs of 0.3, 1, and 3. Labile zinc levels were determined at 24 h p.i. by staining cells with FLZ-3 (C) and ZP-1 (D) by flow cytometry. Data are from at least two independent experiments. Error bars represent means with standard deviations. **, P < 0.01; ****, P < 0.0001.

FIG 2. Labile zinc levels increase in primary epithelial cells. (A) SAECs were infected with RSV at an MOI of 3, and cells were stained with ZP-1 at 24 h p.i. and 48 h p.i. (B) Cell viability was determined by staining the cells with the fixable viability dye eFluor780. (C) Nasal epithelial cells isolated from nasopharyngeal aspirates were stained with ZP-1. Labile zinc levels are presented as median fluorescence intensities (MFI). (D) The cell viability of NPA specimens was determined by staining the cells with the fixable viability dye eFluor780. There were 10 controls and 7 cases. (E) A549 cells were infected with RSV and DENV at MOIs of 0.3 and 5, respectively. Cells were stained with Annexin-V and eFluor780 dye at 24 h p.i. and 48 h p.i. Cell populations represent different phases: healthy, early apoptosis, late apoptosis, and necrosis. Annexin-V signal was detected in FITC filter and eFluor-780 signal was detected in APC-Cy-7 filter. Data are from at least two independent experiments. *, P < 0.05; **, P < 0.01; ns, differences were not significant.

FIG 3. Total zinc content increases in RSV infection. (A to D) A549 cells were infected with RSV at an MOI of 0.3. The total content of the indicated divalent cations was determined by ICP-MS in RSV-infected A549 cells at 48 h p.i. Data are from at least two independent experiments. Error bars represent standard deviations of the means. **, P < 0.01; ****, P < 0.0001.

FIG 4. Knockdown of ZIP1 inhibits RSV infection in A549 cells. (A) A549 cells were treated with siRNA specific to ZIP1, ZIP8, and scrambled siRNA as a control (labeled as NTC [nontargeted control]). ZIP1 and ZIP8 knockdown (KD) was confirmed at the mRNA level by qRT-PCR. (B) A549 cells were treated with ZIP1 and ZIP8 siRNA and later infected with RSV at an MOI of 0.3. A representative viral titer is represented in numbers of PFU per milliliter. (C) RSV titers from two independent experiments from ZIP1 and ZIP8 knockdown conditions. Error bars represent standard deviations of the means. *, P < 0.05; **, P < 0.01.

FIG 5. Zinc supplementation inhibits RSV infection. (A) A549 cells were infected with RSV and DENV at MOIs of 0.3 and 5, respectively, after adsorption of 100 μM added ZnSO4 . RSV and DENV titers were determined at 24 h p.i. (B) SAECs were infected with RSV at an MOI of 3, and 25 μM ZnSO4 was added after infection. RSV titers were determined at 24 h p.i. and are represented as numbers of PFU per milliliter. (C) RSV genome levels were determined under the zinc-supplemented condition at 24 h p.i. (D) A549 cells were infected with RSV at an MOI of 0.3. After adsorption, cells were treated with metal salts as indicated in the text. Viral titers were determined by plaque assay and are represented as percentages. (E) A549 cells were infected with RSV at an MOI of 0.3, and ZnSO4 was added at 2, 4, 8, 12, and 24 h p.i. Supernatants were collected at 24 h p.i. for the 2-, 4-, and 8-h conditions and at 48 h p.i. for the 12- and 24-h conditions. Viral titers are represented as percentages. Data are from at least two independent experiments. Error bars represent standard deviations of the means. **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 6. Zinc chelation enhances RSV infection. (A) A549 cells were infected with RSV and DENV at MOIs of 0.3 and 5, respectively. After adsorption, cells were treated with 0.5 μM TPEN. Viral titers from two independent experiments are represented as percentages. (B) RSV and DENV genome levels were measured using qRT-PCR. Data are from at least three independent experiments. Error bars represent standard deviations of the means. **, P < 0.01; ***, P < 0.001.

FIG 7. Interaction between zinc homeostasis, oxidative stress, and RSV infection. (A) A549 cells were infected with RSV at an MOI of 0.3. ROS levels were measured using the H2DCFDA method. (B) Gene expression were measured for NOX1, CAT, GSTA2, and SOD1 at 48 h p.i. under the RSV infection condition. (C and D) A549 cells were treated with 100 μM ZnSO4 and 0.5 μM TPEN. ROS levels were measured at the indicated time points using H2DCFDA by flow cytometry. The fold changes in ROS levels are displayed for the zinc supplementation and zinc depletion conditions at the indicated time points. Data are from at least two independent experiments, and error bars indicate standard deviations from the means. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 8. Oxidative stress modulates RSV infection. (A and B) A549 cells were infected with RSV at an MOI of 0.3. H2O2 (300 μM) and NAC (10 mM) were added. RSV titers were measured at 24 h p.i. for the H2O2 treatment condition, whereas 24-h p.i. and 48-h p.i. cell supernatants from the NAC treatment were subjected to plaque assay. Viral titers are represented as percentages. Data are from at least two independent experiments, and error bars indicate standard deviations from the means. ***, P < 0.001.