Activation of store-operated calcium entry and mitochondrial respiration by enterovirus 71 is essential for efficient virus replication

Fig 1. EV71 infection induced elevated cytosolic Ca2+ levels that correlated with the progression of viral replication. RD cells were mock-infected (M) or infected with EV71 stock at an MOI of 0.5 (A, B) or 5 (C, D) for the indicated times in the presence of BAPTA (10 µM), EGTA (1.8 mM), or DMSO control. (A, C) Cells were stained with Fluo-8 for 30 min. Fluorescence intensities were quantified relative to the mock-infected controls. (B, D) Cell lysates and supernatants were collected at the indicated times. Total virus titers were determined by TCID50 assay (See Text S1). Data represent mean values from experiments conducted in triplicate, with error bars indicating standard deviations (SDs). ***P < 0.001, **P < 0.01, *P < 0.05 versus the DMSO control.

Fig 2. A specific SOCE blocker, AnCoA4, reduced virus-induced cytosolic Ca2+ levels and viral replication. (A) RD cells were infected with EV71 at an MOI of 0.5 for 6 h in the presence of a DMSO control or 10 and 20 µM AnCoA4. RD cells were mock-infected (M) or infected with EV71 stock at an MOI of 0.5 (B, C) or 5 (D, E) for the indicated times in the presence of 20 µM AnCoA4 or DMSO control. (B, D) Cells were stained with Fluo-8 for 30 min. Fluorescence intensities were quantified relative to the mock-infected controls. (C, E) Cell lysates and supernatants were collected at the indicated time. Total virus titers were determined by the TCID50 assay. (F) Timing of EV71 inhibition by AnCoA4 with the schematic representation. AnCoA4 was added to RD cells at the indicated times relative to viral infection at an MOI of 0.5, with the initiation (0 h) and completion (1 h) of viral adsorption indicated. The bars indicate the periods with AnCoA4 treatment. Viral supernatants and cell lysates were collected at the end of 6 h for all treatment conditions. (I) Whole: −1 to 6 h; (II) entry: −1 to 1 h; (III) early: 1–3 h; (IV) viral replication: 3–6 h. (G) Total virus titers were determined by TCID50…

Fig 3. Both Orai1 and STIM1 are essential during active replication of EV71. HeLa cells were transduced with lentivirus–shScramble, –shOrai1, or –shSTIM1 48 h prior to infection with EV71 at 0.5 MOI for 6 h and 12 h. (A) Cells were stained with Fluo-8 for cytosolic Ca2+ measurement. (B) Cell lysates were harvested, and levels of viral VP1 protein and tubulin (the internal control) were measured by immunoblot analysis. The percentage shown below each lane represents the intensity of viral VP1 relative to that of tubulin. (C) Total virus titers were determined by TCID50. The experiments were conducted in triplicate and are shown as means ± SDs. ***P < 0.001, **P < 0.01, *P < 0.05 versus the scramble control.

Fig 4. EV71 infection and viral 2B expression induced enhanced colocalization of STIM1 and Orai1. (A) HeLa-STIM1-Orai1 cells coexpressing EGFP–STIM1 (green) and mOrange–Orai1 (red) were mock-infected or infected with EV71 at an MOI of 1 for 9 h in the absence or presence of AnCoA4 at 20 µM. Treatment with 5 µM thapsigargin for 15 min served as a positive control for SOCE activation. (B) HeLa-STIM1-Orai1 cells were also transfected with pCMV-Flag-2B (2B) plasmid for 48 h. EV71 VP1 (A) or 2B (B) was detected using the anti-EV71 primary Ab or anti-Flag Ab, respectively, followed by the Cy5-labeled secondary antibody (blue). Colocalization of EGFP–STIM1 and mOrange-Orai1 is presented in yellow. Cell nuclei were stained with DAPI (white). (C, D) The colocalization between EGFP–STIM1 and mOrange-Orai1 was measured using Mander’s coefficients. Each bar represents the mean Mander’s coefficient for a specific experimental condition, reflecting the degree of colocalization between Orai1 and STIM1. Images were acquired using a Zeiss LSM880 confocal microscope. The scale bar represents 5 µM. ***P < 0.001,**P < 0.01, *P < 0.05 versus the mock-infected or EV71-infected group.

Fig 5. Transcriptome profiling identified mitochondrial ETC genes differentially expressed during EV71 infection. (A) Heatmap of differentially expressed genes between the DMSO- and AnCoA4-treated groups, both in the context of EV71 infection at an MOI of 0.5 for 6 h in RD cells. The numbers for each gene indicated the change folds. (B) RD cells were infected with EV71 at an MOI of 0.5 for 6 h or mock infected. The mock-infected cells or EV-infected cells were treated with 20 µM AnCoA4 or DMSO. Relative RNA levels of representative components of ETC complexes, as indicated, were determined through RT-qPCR using total RNA prepared under each condition and shown as levels relative to those of mock-infected controls. RNA level was normalized with an internal control GAPDH. ***P < 0.001,**P < 0.01, *P < 0.05 versus the DMSO control or EV71-infected group.

Fig 6. Expression of 2B and EV71 infection induces transcriptional upregulation of mitochondrial ETC genes through the SOCE mechanism. (A) HeLa cells were transfected with either the pFLAG-CMV-2 (empty) plasmid or the pCMV-FLAG-2B (2B) plasmid in a 6 cm culture dish for 24 h. The cells were trypsinized and divided into wells in a 24-well culture plate to ensure consistent transfection efficiency across wells, and cells were allowed to incubate for an additional 16 h. For the empty controls, the media were replaced with DMSO-containing media and incubated for 8 h. In the case of cells transfected with pCMV-FLAG-2B, the media were replaced with DMSO, 10 µM BAPTA, 1.8 mM EGTA, or 20 µM AnCoA4-containing media, as indicated, followed by an 8 h incubation. The cells were then stained with the cytosolic Ca2+ fluorescent dye Fluo-8. (B) HeLa cells were transfected with either the empty or the 2B-expressing plasmids for 24 h in a 6 cm dish, followed by transferring to wells of a 24-well culture plate for 16 h. They were subsequently incubated with either DMSO or 20 µM AnCoA4 for 8 h. (A, B) 2B expression levels were validated by RT-qPCR for each condition as described in Text S1. HeLa cells…

Fig 7. EV71-infected cells displayed increased mitochondrial respiration and ATP production through the SOCE machinery. (A) Diagram illustrating the oxygen consumption rate (OCR) measured for the parameters including basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and non-mitochondrial oxygen consumption of ETC function, using the XFe24 Seahorse analyzer; this diagram was reproduced with permission. (B) HeLa cells were mock-infected or infected with EV71 (MOI = 5) for 6 h in the absence or presence of AnCoA4 at 20 µM. Oligomycin, FCCP, and antimycin A + rotenone were injected at the times indicated in (A). (C) The six parameters were analyzed following the manufacturer’s instructions and are presented as percentages relative to the mock-infected cells. (D) Mitochondria were isolated from HeLa cells for the complex I activity assay and shown as levels relative to those of mock-infected controls. (E) Cellular ATP activity was measured by a luminescence assay and reported as levels relative to those of mock-infected controls. (F, G) HeLa cells were infected with EV71 at an MOI of 0.5 with oligomycin treated at the indicated concentrations or…

Fig 8. EV71 infections increased the mitochondrial Ca2+ influx that contributed to viral replication and ATP production at the mid-stage of the virus life cycle. (A) HeLa cells were treated with 10 µM RR, 20 µM AnCoA4, or DMSO control during EV71 infection at an MOI of 0.5 for 6 h and 12 h and were then stained with Rhod-2. TG served as a positive control for Rhod-2 stain. The relative Rhod-2 fluorescence intensity of each condition was quantified relative to that of a mock-infected control (M) and normalized by the number of cells stained with DAPI. (B) HeLa cells were transfected with either the empty or the 2B-expressing plasmid for 24 h in a 6 cm dish, followed by transferring to wells of a 24-well culture plate for 16 h. The media were replaced with DMSO, 10 µM RR, or 20 µM AnCoA4-containing media for 8 h and then stained with Rhod-2. 2B expression levels were validated by RT-qPCR for each condition as described in Text S1. (C) HeLa cells were infected with EV71 at an MOI of 5 for 6 h or mock-infected and treated with 10 µM RR or DMSO. Cellular ATP activity was measured and reported as levels relative to those of the mock-infected control. HeLa cells were infected with EV71, CVA16,…

Fig 9. The SOCE inhibitor AnCoA4 attenuated EV71-induced apoptosis and the viral release at the late stage of infection. (A) RD cells were mock-infected or infected with EV71 at an MOI of 5 for 12 h. The infected cells were treated with the indicated concentrations of AnCoA4 or DMSO control. (B) EV71 was used to infect RD cells at MOIs of 1, 2, and 5 for 12 h, and cells were treated with 10 µM AnCoA4 or DMSO control. Cell viability was measured using the MTS assay and compared with that of mock-infected controls. (C) RD cells were mock-infected or infected with EV71 at an MOI of 1 for 12 h and treated with 20 µM AnCoA4 during infection or DMSO control. Caspase-3, -8, and -9 activities were measured using the caspase fluorometric assays. RD cells were infected with EV71 at an MOI of 0.5 for 12 h and treated with AnCoA4 (D) or RR (F), both at 10 and 20 µM, during the infection period. At 12 h p.i., the supernatant was collected for the extracellular virus (ExV), and cells were trypsinized for preparing the intracellular virus (InV). The titers of ExVs and InVs in each group were determined using the TCID50 assay (D, F). For each condition, the relative ratio of the ExV titer over the…

Fig 10. EV71 replication and virus-upregulated ETC RNA were reduced by AnCoA4 in the mouse intestinal organoid model. (A) Schematic representation of the three-dimensional mouse intestinal organoid culture. (B) Mouse intestinal organoid cultures seeded in Xfe 24-well dishes were inoculated with 200 µL EV71 MP4 strain (4 × 108 TCID50/mL) for 48 h and treated with 20 µM AnCoA4 during the infection period or DMSO control. An immunofluorescence assay was conducted, with viral VP1 antigen detected using the mouse anti-EV71 Ab (green) and the nucleus stained with DAPI (blue). (C) The relative VP1 positive (+) antigen expression of MP4-infected, DMSO-treated, and MP4-infected, AnCoA4-treated cells was quantified relative to the number of cells stained with DAPI. Total RNA was prepared, and viral RNA (D) and the indicated mitochondrial ETC RNA (E) were measured through RT-qPCR and presented as levels relative to those of the mock-infected controls (%). Data are presented as means ± SDs. ***P < 0.001, **P < 0.01, *P < 0.05 versus the DMSO control.

Fig 11. Proposed mechanism through which EV71-induced cytosolic Ca2+ elevation affects the virus life cycle through SOCE activation and mitochondrial Ca2+ influx. EV71 2B caused ER Ca2+ depletion, resulting in SOCE activation. At 6 h p.i., cytosolic Ca2+ levels increased, causing a moderate elevation in mitochondrial Ca2+ and subsequent increases in mitochondrial electron transport chain complex (I through IV) activity and ATP generation for viral replication. At 12 h p.i., overloading of mitochondrial Ca2+ promoted viral release through apoptotic cell death triggered by the intrinsic apoptosis pathway. AnCoA4 and RR reduced the levels of cytosolic Ca2+ and mitochondrial Ca2+, reducing viral replication and virus release, respectively. Oligomycin targets the mitochondrial complex V (ATP synthase), leading to inhibition of viral replication. RR = ruthenium red. MCU = mitochondrial Ca2+ uniporter.