Enterovirus 71 induces mitochondrial reactive oxygen species generation that is required for efficient replication

Abstract

Redox homeostasis is an important host factor determining the outcome of infectious disease. Enterovirus 71 (EV71) infection has become an important endemic disease in Southeast Asia and China. We have previously shown that oxidative stress promotes viral replication, and progeny virus induces oxidative stress in host cells. The detailed mechanism for reactive oxygen species (ROS) generation in infected cells remains elusive. In the current study, we demonstrate that mitochondria were a major ROS source in EV71-infected cells. Mitochondria in productively infected cells underwent morphologic changes and exhibited functional anomalies, such as a decrease in mitochondrial electrochemical potential ΔΨ(m) and an increase in oligomycin-insensitive oxygen consumption. Respiratory control ratio of mitochondria from infected cells was significantly lower than that of normal cells. The total adenine nucleotide pool and ATP content of EV71-infected cells significantly diminished. However, there appeared to be a compensatory increase in mitochondrial mass. Treatment with mito-TEMPO reduced eIF2α phosphorylation and viral replication, suggesting that mitochondrial ROS act to promote viral replication. It is plausible that EV71 infection induces mitochondrial ROS generation, which is essential to viral replication, at the sacrifice of efficient energy production, and that infected cells up-regulate biogenesis of mitochondria to compensate for their functional defect.

Figure 1. EV71 infection induces ROS in neural cells in a time-dependent manner.

SF268 cells were infected with EV71 at m.o.i. of 1.25, 1.5 and 2 for 0, 12, 24, 36, 48, 60 and 72 hr, and were subject to H₂DCFDA staining and flow cytometric analysis. (A) Representative histograms of cell counts (counts) vs. DCF fluorescence (FL1-H) for cells infected at an m.o.i. of 1.25 at indicated times are shown. (B) The mean fluorescence intensity (MFI) of DCF of infected cells is expressed as fold change relative to that of uninfected cells. The results are presented as mean±SD of three separate experiments.

Figure 2. EV71 infection causes mitochondrial ROS production.

(A) SF268 cells were mock- (A) or infected with (B) EV71 at an m.o.i. of 1.25 for 54 hr, and were subject to H₂DCFDA and Mitotracker Red staining and confocal microscopic examination. The MitoSOX-stained mitochondria (a), DCF-stained ROS generation sites (b), and Hoechst 33342-stained nuclei (c) are shown. The corresponding images are overlaid (d). The photographs shown here are representative of three experiments. Scale bar, 20 µm. (C) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25 for 48 hr, and were subject to MitoSOX Red staining and flow cytometric analysis. (a) Representative histograms of cell counts (counts) vs. MitoSOX fluorescence (FL2-H) for un- (left panel) and infected cells (right panel) are shown. (b) The mean fluorescence intensity (MFI) of MitoSOX of mock- (−) and infected (+) cells is expressed as the percentage of that of uninfected cells. The results are presented as mean±SD, n = 3. *p<0.05 vs. uninfected cells.

Figure 3. Mitochondrial electron transport-dependent ROS generation.

SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25, and treated without or with apocyanin, rotenone or antimycin A. Forty-eight hours later, cells were stained with H₂DCFDA and analyzed by flow cytometry. The MFI of DCF of infected cells is expressed as the percentage of that of uninfected cells. The results are presented as mean±SD, n = 3. *p<0.05 vs. untreated, uninfected cells; ^#p<0.05, drug-treated vs. untreated cells.

Figure 4. EV71 infection results in morphological changes in mitochondria.

SF268 cells were mock- (A & C) or infected with (B, D–G) EV71 at an m.o.i. of 1.25 for 48 hr, and processed for electron microscopic examination. The mock-infected cells had typical nucleus (N) and mitochondria (M). In EV71-infected cells, a number of mitochondria underwent changes in morphology, characterized by deranged cristae (D & E). The developing viral replication site (RS) was lined with ribosomes and was in proximity to mitochondria (D). Numerous single or double membrane-bound vesicles (MV) developed in EV71-infected cells, and some contained virus particles (VP). For A & B, bar represents 5 µm; for F, bar represents 1 µm; for C, D, E & G, bar represents 0.2 µm.

Figure 5. EV71 infection causes decline in ΔΨ_m.

SF268 cells were mock- (A) or infected with (B) EV71 at an m.o.i. of 1.25 for 48 hr. Cell were stained with JC-1 and Hoechst 33342 dyes, and examined by confocal microscopy. Intracellular distribution of JC-1 J-aggregate (a) and monomer (b) is indicative of ΔΨ_m in cells. Nuclei of these cells are shown (c). The corresponding images are overlaid (d). The photographs shown here are representative of three experiments. Scale bar, 20 µm. (C) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25 for indicated times, and were subject to JC-1 staining and flow cytometric analysis. The ratio of MRI of FL2 channel to that of FL1 channel (FL2/FL1) was calculated, and is expressed relative to that of uninfected cells. Results are mean ± SD, n = 3. *p<0.05 vs. uninfected cells.

Figure 6. EV71 infection-induced oxygen consumption is associated with a reduction in respiratory efficiency.

(A) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25 for indicated times. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (10⁻² µg O₂/5×10⁵ cells/min) was calculated accordingly. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells. (B) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25, and were treated without or with oligomycin. Oxygen concentration was assayed with Clark oxygen electrode, and oxygen consumption rate (10⁻² µg O₂/5×10⁵ cells/min) was calculated. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells; ^#p<0.05, oligomycin-treated vs. untreated cells. (C) Oligomycin-sensitive oxygen consumption rate was calculated as the difference in the absence and presence of oligomycin. (D & E) The oxygen consumption was measured as described in (A) and (B). Data were normalized to the relative mitochondrial mass unit (MMU) of control and infected cells. Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells. (F) Oligomycin-sensitive oxygen consumption rate was calculated as described in (C), and normalized to the relative mitochondrial mass unit (MMU) of control and infected cells. (G–I) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25 for 48 hr, and mitochondria were isolated and assayed for oxygen consumption rates (10⁻¹ µg O₂/mg mitochondrial protein/min) during state 3 (G) and 4_o (H) respiration as described in Materials and Methods. RCRs were calculated accordingly, and are shown (I). Results are mean ± SD, n = 6. *p<0.05 vs. uninfected cells.

Figure 7. Mitochondrial mass increases and expression of mitochondrial proteins changes in response to EV71 infection.

(A) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25 for 48 hr, and were subject to Mitotracker dye staining and flow cytometric analysis as described in Materials and Methods. The MFI of the stained cells is expressed relative to that of control cells. Results are mean ± SD, n = 3. *p<0.05 vs. uninfected cells. (B & C) Cells were un- or infected under the similar condition, and mitochondria were isolated for SDS-PAGE electrophoresis and silver staining (B). In the silver-stained gel, the leftmost lane corresponds to protein markers with respective molecular weights indicated alongside the bands. (C) Cells similarly infected were harvested for western blotting with indicated antibodies. A representative experiment out of three is shown here.

Figure 8. Levels of ATP, ADP and AMP in EV71-infected cells.

SF268 cells were mock- (Con) or infected (Infected) with EV71 at an m.o.i. of 1.25 for 48 hr, and were harvested for UPLC-based analyses of ATP, ADP and AMP. These adenine nucleotides are normalized to cellular protein content. Levels of ATP, ADP and AMP (A) and total adenine nucleotides (B) are shown. Results are mean ± SD, n = 3. *p<0.05 vs. uninfected cells.

Figure 9. Mitochondrial ROS are essential to EV71 replication.

(A) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25, and treated without (Con) or with indicated concentrations of Mito-TEMPO. Forty-eight hours later, cells were subject to MitoSOX Red staining and flow cytometric analysis. The mean fluorescence intensity (MFI) of MitoSOX of mock- and infected cells is expressed as the percentage of that of uninfected cells. The results are presented as mean ± SD, n = 3. *p<0.05 vs. infected Con group. (B) SF268 cells were mock- or infected with EV71 at an m.o.i. of 1.25, and treated without or with 200 µM of Mito-TEMPO. Forty-eight hours later, cells were harvested for western blotting with antibodies to phosphorylated eIF2α and total eIF2α, viral protein 3D, and actin. A representative experiment out of three is shown here. (C) SF268 cells were mock- (−) or infected (+) with EV71 at an m.o.i. of 1.25, and treated without (Con) or with 200 µM of Mito-TEMPO. Forty-eight hours later, cells were analyzed for levels of EV71 genomic RNA. The results are presented as means ± SD n = 3. *p<0.05 vs. infected Con group.