Coxsackievirus B Escapes the Infected Cell in Ejected Mitophagosomes

Abstract

Coxsackievirus B (CVB) is a common enterovirus that can cause various systemic inflammatory diseases. Because CVB lacks an envelope, it has been thought to be inherently cytolytic, wherein CVB can escape from the infected host cell only by causing it to rupture. In recent years, however, we and others have observed that various naked viruses, such as CVB, can trigger the release of infectious extracellular microvesicles (EMVs) that contain viral material. This mode of cellular escape has been suggested to allow the virus to be masked from the adaptive immune system. Additionally, we have previously reported that these viral EMVs have LC3, suggesting that they originated from autophagosomes. We now report that CVB-infected cells trigger DRP1-mediated fragmentation of mitochondria, which is a precursor to autophagic mitochondrial elimination (mitophagy). However, rather than being degraded by lysosomes, mitochondrion-containing autophagosomes are released from the cell. We believe that CVB localizes to mitochondria, induces mitophagy, and subsequently disseminates from the cell in an autophagosome-bound mitochondrion-virus complex. Suppressing the mitophagy pathway in HL-1 cardiomyocytes with either small interfering RNA (siRNA) or Mdivi-1 caused marked reduction in virus production. The findings in this study suggest that CVB subverts mitophagy machinery to support viral dissemination in released EMVs.IMPORTANCE Coxsackievirus B (CVB) can cause a number of life-threatening inflammatory diseases. Though CVB is well known to disseminate via cytolysis, recent reports have revealed a second pathway in which CVB can become encapsulated in host membrane components to escape the cell in an exosome-like particle. Here we report that these membrane-bound structures derive from mitophagosomes. Blocking various steps in the mitophagy pathway reduced levels of intracellular and extracellular virus. Not only does this study reveal a novel mechanism of picornaviral dissemination, but also it sheds light on new therapeutic targets to treat CVB and potentially other picornaviral infections.

Keywords: autophagy; coxsackievirus; dissemination; mitochondria; mitophagy.

FIG 1

Impaired autophagy results in reduced viral dissemination. Wild-type (WT) and ATG5 knockout (ATG5KO) mouse embryonic fibroblasts (MEFs) were infected with eGFP-expressing coxsackievirus B (eGFP-CVB) at a multiplicity of infection of 10 (MOI10). (A) Fluorescence microscopy of infected MEFs at 8 h and 24 h postinfection (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. (E) Phase-contrast and fluorescence microscopy images of MEFs infected with timer protein-expressing CVB. Scale bars represent 30 μm.

FIG 2

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.

FIG 3

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.

FIG 4

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.

FIG 5

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.

FIG 6

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.

FIG 7

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.

FIG 8

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.

FIG 9

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.

FIG 10

Schematic of mitophagy subversion by CVB. (A) Upon infection, CVB virions localize to mitochondria and trigger DRP1-mediated mitochondrial fission. The resultant mitochondrial fragments undergo autophagic engulfment. Rather than undergoing lysosomal degradation, these mitophagosomes containing both mitochondrial components and CVB virions are instead ejected from the cell as viral EMVs. Accumulation of mitophagosomes may also cause them to fuse together as megaphagosomes prior to becoming released. (B) Blocking DRP1 with Mdivi-1 (or siDRP1) inhibits mitochondrial fragmentation and subsequent initiation of mitophagy. This results in impaired EMV biogenesis, causing CVB virions to accumulate in the cell. (C) Due to a lack in dissemination, the virus may rely more on cytolytic cell death resulting in the release of free virions. In vivo, this mode of viral release would leave the virus more susceptible to neutralizing antibodies.