Human Enterovirus Group B Viruses Rely on Vimentin Dynamics for Efficient Processing of Viral Nonstructural Proteins

Figures

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  FIG 1. Human enterovirus infection induces vimentin-enwrapped dsRNA-harboring compartment to the perinuclear location in A549 cells. A549 cells were fixed, immunolabeled, and visualized with confocal microscopy. (A) Images from single sections showing vimentin (green) and virus capsid (magenta) in cells after CVB3 (5 h p.i.) infection. (B) Vimentin (green) and tubulin (magenta) network in noninfected (ctrl) and CVB3-infected (5 h p.i.) cells. Infected cells are marked with asterisks. (C) Images from single sections showing vimentin (green) and virus capsid (magenta) in cells after EV1 (6 h p.i.), CVB1 (6 h p.i.), and CVA9 (5 h p.i.) infections. Noninfected cells are marked with asterisks. (D) Projection of Z-sections showing dsRNA (green) and vimentin (magenta). Orthogonal sections providing a view of these structures in three dimensions after CVB3 infection (5 h p.i.). (E) Images of single sections showing vimentin structure formation from 2.5 h to 6 h p.i. Cell boundaries were drawn to visualize the state of cell detachment.

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  FIG 2. Appearances of VP1 and dsRNA coincide with vimentin rearrangements during CVB3 infection. A549 cells were fixed, immunolabeled, and visualized with confocal microscopy. (A) Single-section images showing vimentin and VP1 (capsid) at different time points p.i. (B) Quantifications of confocal images taken at different time points during CVB3 infection. The results shown here are representations of at least three independent experiments. For the quantifications, approximately 200 cells altogether from two to three replicates were analyzed (±SEM). Scale bars, 20 μm.

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  FIG 3. Viral protein synthesis is essential for vimentin cage formation. (A, top) Schematic illustration showing the principle of neutral red viruses. A549 cells were infected with neutral red-CVB3 exposed to light treatment at different time points, and the presence of virus-induced vimentin structures was visualized from single-section confocal images and quantitated. For the quantifications, approximately 50 cells per sample from three replicates were analyzed. (B) Single-section confocal images illustrating the effect of UV-inactivated EV1 on A549 cells at 6 h p.i. Cells were immunolabeled for capsid (green) and vimentin (magenta). (C) Graphs showing the results of the CPE experiment in CVB3-infected A549 cells after differential treatments with either cycloheximide (200 μg/ml) or puromycin (100 μg/ml). Drugs were added to the cells at different time points p.i. and left until the end of the experiment (8 h p.i.). Control cells were normalized to 100%. Data are representative of at least two separate experiments with three replicate samples from each. (D) Single-section confocal images visualizing vimentin (green) and virus capsids (red) in CVB3-infected (5 h p.i.) A549 cells with…

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  FIG 4. IDPN treatment delays virus-induced cell death without compromising the production of progeny viruses. (A) Graph showing the effect of IDPN treatment on A549 cell viability. Results are representative of two replicates. (B) Single-section images showing vimentin distribution after 5.5 h of 1.5% IDPN treatment. Results are representative of at least three separate experiments. (C) Graph showing cell viability (ATP) in CVB3-infected A549 cells with and without IDPN treatment (1.5%). Drug was added together with the virus and kept until the end of the experiment. Results are representative of at least two separate experiments with three replicate samples from each. (D) Endpoint titration of progeny viruses produced after 6 h of CVB3 infection in A549 cells with or without IDPN treatment. Results are representative of two independent experiments. (E) RT-qPCR from cells infected with CVB3 for 1, 3, 4, or 5 h with or without IDPN treatment. Virus (8.86 × 107 PFU/ml) was bound on cells on ice for 1 h. After washing excess virus away, the infection was allowed to proceed for the indicated time. IDPN was added after ice binding and left until the end of the experiment. (F) Single-section…

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  FIG 5. Vimentin cage preferentially hosts nonstructural proteins. (A and B) Single sections showing location of 3D (magenta) (A) or 2A (magenta) (B) in the perinuclear area and vimentin (green) in control or CVB3-infected cells with or without IDPN treatment. Virus (4.43 × 108 PFU/ml) was bound on ice for 1 h. After washing the excess virus away, infection was allowed to proceed for 5.5 h. IDPN was added after ice binding and left until the end of the experiment. (C) Single sections showing the location of VP1 diffusely in the cytoplasm in CVB3-infected cells with or without IDPN treatment. Infection was carried out as described for panel B. Scale bars, 20 μm. Representative images from at least three separate experiments are shown. (D and E) Single-section confocal images illustrating the effect of IDPN on ER (PDI) (5.5. h p.i) (D) and Golgi (GM130) (5.5 h p.i) (E). Scale bar, 20 μm. The images are representative of at least two separate experiments.

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  FIG 6. Inhibition of vimentin dynamics affects the levels and activity of nonstructural proteins. (A) Graphs showing quantifications of Western blots where levels of VP1, 2A, 3C, and 3D were detected in CVB3-infected A549 cells with or without IDPN treatment. Virus (4.43 × 108 PFU/ml) was bound on ice for 1 h. After washing the excess virus away, infection was allowed to proceed for 5.5 h. IDPN was added after ice binding and left until the end of the experiment. Band intensities were quantified using Image J, and the quantifications were done from at least three separate experiments. (B) Western blots were immunolabeled with antibodies against PABP, G3BP1, VP1, and GAPDH. The arrowhead indicates CVB3 induced cleavage product. Results are representative of at least two separate experiments. (C and D) Graphs showing the results of the viability measurement (C) and caspase activity (D) per viable cell of A549 cells treated with Z-VAD-fmk (200 μM) or IDPN (1.5%) with or without CVB3 infection. Drugs were added to the cells together with the virus and left until the end of the experiment (10 and 24 h p.i.). Graphs show results from three independent experiments. (E) Western blots were…

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  FIG 7. Vimentin dynamics affect the synthesis of nonstructural proteins rather than their degradation. (A) Western blot of A549 cells infected with CVB3 for 3, 4, 5, or 6 h with or without IDPN treatment. Virus (4.43 × 108 PFU/ml) was bound on ice for 1 h. After washing the excess virus away, infection was allowed to proceed for the indicated time. IDPN was added after ice binding and left until the end of the experiment. eIF4G, 3D, 2A, and VP1 were visualized using antibodies against the proteins. Shown is a representative image of two replicates. (B) Western blot showing the effect of 1.5% IDPN, 7 μM bortezomib, 200 μM calpain inhibitor 1, 100 μg/ml puromycin, 50 μM VER-155008, or 0.1 μM geldanamycin on CVB3 infection. Virus (4.43 × 108 PFU/ml) was bound on ice for 1 h. After washing the excess virus away, infection was allowed to proceed for 5.5 h. Other drugs were added after ice binding except calpain inhibitor, bortezomib, and puromycin, which were added at 2 h p.i. All drugs were left until the end of the experiment. Visualization of VP1 and P1 is on the left. Merged image of 2A and VP1 labeling is on the right. Results are representative of at least two separate experiments.

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  FIG 8. Summary. Viral protein synthesis during enterovirus infection induces formation of a vimentin-enwrapped perinuclear compartment harboring the viral nonstructural proteins. Inhibition of vimentin rearrangements leads to scattered distribution of nonstructural proteins and their lower expression and activity without affecting the structural proteins and viral progeny production. Stars indicate the magnitude of the phenomenon. NS-proteins, nonstructural proteins.