EV-D68 exploits clathrin-mediated endocytosis and compensatory macropinocytosis for cellular entry

Figures

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  Fig 1. EV-D68 TW-02795-2014 infection is dependent on sialic acid and inhibited by endocytic inhibitors. (A) Schematic illustration of neuraminidase treatment protocol. (B) RD cells were pre-treated with 50 mU of neuraminidase for 1 h and subsequently infected with EV-D68 at an MOI of 10. The cells were harvested at 6 h p.i., and viral protein expression was analyzed by immunoblotting with specific antibodies against VP1. Quantification of signal intensity is shown in the right panel. (C–D) RD cells were pre-treated with SNA or MAL-I and infected with EV-D68 at an MOI of 1. Viral RNA levels were harvested at 6 h p.i. for qRT-PCR analysis. (E) Schematic illustration of inhibitor treatment. The cells were pre-treated with chemical inhibitors in DMEM for 2 h (−3 to −1 h p.i.) at 37°C. EV-D68 (MOI = 1) was incubated with cells in inhibitor-containing DMEM for 1 h at 33°C. The cells were incubated in E2 for 6 h at 33°C. The viral protein, viral RNA, and viral production were analyzed with immunoblotting (F), qRT-PCR (G), and plaque assay (H), respectively. Immunoblotting images represent three independent replicates. (I) RD cells were pre-treated with selected chemical inhibitors in DMEM for…

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  Fig 2. Clathrin and EV-D68 VP1 co-localization during early stages of infection and disruption by chemical inhibitor. Subcellular localization of EV-D68 VP1 (red) and clathrin (green), as well as their co-localization, was analyzed by immunofluorescence confocal microscopy using antibodies specific to clathrin heavy chain and VP1. (A) EV-D68 was incubated with RD cells in DMEM at an MOI of 200 for 1 h at 33°C. More than 30 cells per group were quantified using Zeiss Zen software. (B) EV-D68 strain was incubated with RD cells in DMEM at an MOI of 100 for 15 min at 4°C. The cells were then transferred to 33°C to facilitate viral entry. (C) RD cells were treated with CPZ after virus adsorption and incubated with EV-D68 at 33°C for 45 min. More than 30 cells per group were quantified using Zeiss Zen software, with median values indicated. Data are presented as the median for each group. Statistical comparisons were performed using the Mann–Whitney U-test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

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  Fig 3. Disruption of clathrin-mediated endocytosis through siRNA knockdown or dominant-negative Eps15 overexpression did not affect EV-D68 infection in RD cells. (A) The siRNA of clathrin and AP2 was transfected in RD cells for 3 days, and the knockdown efficiency was measured by qRT-PCR. (B) Transferrin-FITC (green) and Hoechst (blue) were added to transfected cells and incubated at 33°C for 1 h, followed by fixation with 4% PFA. The FITC intensity in each cell was quantified using Investigator software, and statistical analysis was performed using the Mann–Whitney U-test. (C–E) EV-D68 was incubated with cells in DMEM at an MOI of 1 for 1 h at 33°C. Inhibition of viral replication in the context of expression of viral protein, viral RNA, and viral titers in RD cells transfected with siCHC and siAP2M1. The viral protein, viral RNA, and viral production were analyzed with immunoblotting (C), qRT-PCR (D), and plaque assay (E), respectively. Immunoblotting images represent three independent replicates. Mean raw virus titers are indicated. (F) siCHC or siAP2M1 was transfected in RD cells for 3 days. Cells were incubated with EV-D68 (MOI 1) for 1 h at 4°C, after which the inoculum was…

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  Fig 4. Inhibitory effects of chemical inhibitors and CME component modulation on EV-D68 infection in A549 cells. (A) Schematic illustration of inhibitor treatment. A549 cells were treated with chemical inhibitors using the same conditions as applied to RD cells in Fig. 1D. The viral protein, viral RNA, and viral production were analyzed with immunoblotting (B), qRT-PCR (C), and plaque assay (D), respectively. Immunoblotting images represent three independent replicates. (E–H) Knockdown of clathrin and AP-2 in EV-D68 infection in A549 cells. The siRNA of clathrin and AP2 was transfected in A549 cells for 3 days, and knockdown efficiency was measured by qRT-PCR (E). The cells were infected with EV-D68 at an MOI of 1 in DMEM for 1 h at 33°C, followed by a 6-h incubation in E2. Viral protein, RNA, and production were assessed by immunoblotting (F), qRT-PCR (G), and plaque assay (H), respectively. Immunoblotting images represent three independent replicates. Mean raw virus titers are indicated. Data are presented as means ± SD of three replicates; comparisons were carried out using the Student’s t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, no significance).

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  Fig 5. Transmission electron microscopy images of infected cell surface and large membrane compartment-associated EV-D68. Canonically purified and concentrated EV-D68 using 100 kDa MWCO Amicon ultra centrifugal filters was incubated with RD cells in DMEM at an MOI of 7,000 for 1 h at 33°C. (A) Clustering of EV-D68 particles (Area 1, white arrowheads) and membrane protrusion (Area 2, black arrow). The insets display the images at higher magnification. (B) EV-D68 was observed in the junction between the plasma membrane and the large membrane structures (black arrowheads in Areas 1–3) and arrays of viral particles were observed under higher magnification in the insets (white arrowheads in 1 and 2). (C) Internalized EV-D68 particles and associated membrane structures enclosed within intracellular vesicles.

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  Fig 6. Downregulation of clathrin leads to Rac1 upregulation and compensatory activation of macropinocytosis. (A) RD cells were transfected with siRNA for 3 days and then treated with transferrin-FITC or dextran-FITC for 1 h at 33°C. The nucleus was stained with Hoechst, and FITC intensities were quantified using Investigator software from four independent replicates; representative images from one replicate are shown, with median values indicated. (B) Flow cytometric analysis of dextran-FITC uptake in control (SC) and clathrin-depleted (siCHC) cells from four independent experiments, with mean values indicated. (C and D) Rac1 expression level was increased in siCHC-transfected RD and A549 cells. Clathrin and Rac1 expression levels in siRNA-transfected RD (C) and A549 (D) were analyzed by immunoblotting. The images represent the three independent replicates. Data are presented as means ± SD of three replicates; comparisons were carried out using the Student’s t-test and Mann-Whitney U-test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

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  Fig 7. Impact of virus-associated compartments and compensatory macropinocytosis on clathrin-mediated viral entry. (A) Schematics for purification of EV-D68 virus particles from the cultured supernatant with detergent and CsCl gradient ultracentrifugation. EV-D68 VP1 expression in each fraction was examined by immunoblotting to verify the distribution of viral particles across the fractions. Fractions 9–12 (density = 1.2–1.4 g/mL) were pooled, concentrated using an Amicon Ultra centrifugal filter, and examined by TEM (B). (C) siCHC was transfected in RD cells for three days. Crude or CsCl-purified EV-D68 (MOI1) was incubated with cells for 1 h at 4°C. The virus was then removed and the cells were incubated in E2 at 33°C for 1 h. The cells were treated with trypsin prior to harvesting for qRT-PCR analysis. (D) Transfected cells were pretreated with NSC23766 for 1 h at 37°C and then incubated with dextran-FITC for 1 h at 33°C. Images were acquired using a confocal microscope. (E) Flow cytometric analysis of dextran-FITC uptake in control (SC) and clathrin-depleted (siCHC) cells treated with mock (DMSO) or NSC23766. Data are presented as means ± SD of three replicates. (F) Transfected…

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  Fig 8. Proposed model illustrating how EV-D68 exploits both CME and macropinocytosis for cellular entry. Individual EV-D68 particles enter cells via CME following receptor attachment, whereas membrane-associated EV-D68 fails to utilize this pathway. Short-term inhibition of CME using chemical inhibitors does not trigger compensatory cellular responses; however, prolonged inhibition—achieved by overexpression of dominant-negative Eps15 or siRNA-mediated knockdown—leads to enhanced macropinocytosis, thereby facilitating EV-D68 entry.