Coxsackievirus B Escapes the Infected Cell in Ejected Mitophagosomes

Autophagy is essential for the release of virus-induced extracellular microvesicles. WT and ATG5KO MEFs were infected with eGFP-CVB at MOI10. (A) Merged fluorescence and phase-contrast images of infected MEFs 24 h p.i. Scale bars represent 30 μm. Open arrows indicate blebs stemming from the surfaces of infected cells, whereas solid arrows indicated fully detached eGFP⁺ EMVs. (B) EMVs were isolated from C2C12 skeletal myoblasts that were either infected with eGFP-CVB at MOI10 or mock infected. EMVs were stained with a phycoerythrin-conjugated LC3-II antibody and analyzed via flow cytometry. EMVs from mock-infected cells were used for isotype control staining. (C) Western blots of EMVs isolated from HL-1 cells infected with eGFP-CVB at MOI1.

CVB induces fragmentation of mitochondrial networks. HL-1 cells expressing mitochondrion-targeted DsRed (mito-DsRed) were either infected with eGFP-CVB at MOI1 or mock infected. (A) On the left are representative fluorescence microscopy images of a mock-infected cell or an eGFP-CVB-infected cell 24 h p.i. Red, mitochondria; green, viral protein. On the right are thresholded images used for quantifications in panel B. Scale bars represent 20 μm. (B) Quantification of mitochondrial interconnectivity as measured by area/perimeter ratio calculations based on thresholded images of the red channel. $, P < 4.843 × 10⁻⁸, Student t test; n = 25 cells.

CVB-induced viral EMVs contain mitochondrial fragments. Mito-DsRed-expressing HL-1 cells were infected with eGFP-CVB at MOI1. (A) Fluorescence microscopy images of infected cells immunostained for VP1 24 h p.i. Scale bars represent 10 μm. (B) Grayscale images from panel A. Arrows indicate EMVs containing mito-DsRed, viral eGFP, and VP1. Images are presented in grayscale to enhance contrast of all channels. Scale bars represent 10 μm. Further-magnified colored images of EMVs indicated by top and bottom arrows are presented to the right, accompanied by representative line profiling depicting color composition of bottom EMV. (C) Western blots on EMVs isolated from HL-1 cells infected with eGFP-CVB at MOI1 24 h p.i. Two separate membranes are shown. (D) Western blots of cell lysates and EMVs isolated from infected HL-1 cells 24 h p.i. (E) Western blots of washed and nonwashed mitochondria isolated from infected HL-1 cells 24 h p.i. (F) Western blots of cytoplasm (Cyto), outer membrane plus intermembrane space (OM+IMS), and mitoplast fractions from infected HL-1 cells 24 h p.i. Blots were probed for fraction-specific markers to ensure efficient fractionation. These were Rho GDP-dissociation inhibitor 1 (Rho GDI) (cyto), cytochrome c (OM+IMS), and total OXPHOS complexes (mitoplast). From largest to smallest, the bands labeled by the total OXPHOS complex antibody cocktail are as follows: complex 5 (CV), ATP5A; CIII, UQRC2; CIV, MTCO1; CII, SDHB; and CI, NDUFB8. Equal amounts of protein were loaded into each lane.

Silencing DRP1 limits CVB infection. HL-1 cells were treated with siRNA targeting DRP1 (siDRP1) or scrambled RNA (siSCRAMBLE) and subsequently infected with eGFP-CVB at MOI1. (A) Fluorescence microscopy of infected HL-1 cells at 8 h and 24 h p.i. Phase-contrast images show similar cell numbers at 24 h p.i. Scale bars represent 200 μm. (B) Extracellular viral titers of infected cells at 8 h and 24 h p.i. as measured by plaque assay. *, P < 0.05, Student t test; n = 3. (C) Western blots of infected cells at 0 h, 8 h, and 24 h p.i. (D) Densitometric quantification of Western blots in panel C. A representative Western blot is shown. **, P < 0.01, Student t test; n = 3.

Mdivi-1 treatment reduces CVB infection. A 50 μM concentration of Mdivi-1 or an equal volume of DMSO (vehicle control) was added to HL-1 cells 40 min before infection with eGFP-CVB at MOI1 (51). Mdivi-1 was maintained in medium throughout the infection time course. (A) Fluorescence microscopy of infected HL-1 cells at 8 h and 24 h p.i. Phase-contrast images show similar cell numbers at 24 h p.i. Scale bars represent 200 μm. (B) Extracellular viral titers of infected cells at 8 h and 24 h p.i. as measured by plaque assay. *, P < 0.05, Student t test; n = 3. (C) Western blots of infected cells at 0 h, 8 h, and 24 h p.i. (D) Densitometric quantification of Western blots in panel C. A representative Western blot is shown. *, P < 0.01, Student t test; n = 3.

Blocking mitophagy suppresses mitochondrial and viral secretion into EMVs. Shown are Western blots of EMVs isolated from siDRP1-treated (A), Mdivi-1-treated (B), or siOPTN-treated (C) HL-1 cells infected with eGFP-CVB at MOI1 24 h p.i. Untreated cells were given respective controls.

Silencing optineurin reduces CVB infection. HL-1 cells were treated with siRNA targeting optineurin (siOPTN) or siSCRAMBLE and subsequently infected with eGFP-CVB at MOI1. (A) Fluorescence microscopy of infected HL-1 cells at 8 h and 24 h p.i. Phase-contrast images show similar cell numbers at 24 h p.i. Scale bars represent 200 μm. (B) Extracellular viral titers of infected cells at 8 h and 24 h p.i. as measured by plaque assay. **, P < 0.01, Student t test; n = 3. (C) Western blots of infected cells at 0 h, 8 h, and 24 h p.i. (D) Densitometric quantification of Western blots in panel C. A representative Western blot is shown. *, P < 0.05, Student t test; n = 3.

Blocking mitophagy does not impair viral replication. HeLa cervical cancer cells were transfected with either siDRP1, siOPTN, or siSCRAMBLE. Cells were then infected with eGFP-CVB at MOI0.01. Culture media and cell lysates were isolated at various time points. (A) Western blots of infected cells at 4 h, 5 h, 6 h, 7 h, and 8 h following treatment with either siSCRAMBLE or siDRP1. (B) Western blots of infected cells at 4 h, 5 h, 6 h, 7 h, and 8 h following treatment with either siSCRAMBLE or siOPTN. (C) Western blots of infected cells at 24 h p.i. following treatment with either siSCRAMBLE, siDRP1, or siOPTN. (D) Extracellular viral titers of infected cells at 4 h, 5 h, 6 h, 7 h, 8 h, and 24 h p.i. as measured by plaque assay.