Fig 2: Hypothetical model of TBK1 function and CVB infection.CVB has previously been shown to rely on autophagy to promote viral replication. Here, we describe the anti-viral role of the phosphor-kinase TBK1 in autophagic elimination of CVB. (1) Phagophore initiation is by the recruitment and assembly of autophagic machinery. Among the key machinery components, ATG8 family members including LC3, GABARAPL1, and GABARAPL2 regulate phagophore elongation. ATG8 proteins coordinate cargo selection (such as damaged mitochondria and pathogens like CVB) with autophagy adaptors, p62 and Optineurin. TBK1 is known to directly phosphorylate p62 and Optineurin for efficient cargo selection. (2) Upon closure of the phagophore, the autophagosome is formed and fuses with the (3) lysosome to form the autolysosome. Here, TBK1 phosphorylates LC3 and GABARAPL2 to inhibit premature shedding of the autophagosome and promote flux. (4a) Upon fusion, the inner membrane space of the autolysosome undergoes acidification and digestive enzymes are activated to (5) degrade its contents. (4b) However, in the absence of TBK1, the autophagosome subverts fusion with the lysosomes and is released from the intracellular space to the extracellular space as an extracellular vesicle (EV) containing infectious CVB and mitochondrial fragments.

Fig 3: CVB infection increases in a genetic knockout of TBK1.Wild-type (TBK1+/+), heterozygous knockout (TBK1+/-), and homozygous knockout (TBK1-/-), mouse embryonic fibroblasts (MEFs) were infected with eGFP-expressing coxsackievirus B at a multiplicity of infection of 10 (MOI 10). (A) Fluorescence microscopy of infected MEFs at 24 h post-infection (p.i.). Phase contrast images show similar cell density at 24 h p.i. Scale bars represent 100 μm. (B) Extracellular viral titers of infected cells at 24 h as measured by plaque assay. ***, p < 0.001. Student t test n = 3. (C) Diameter of viral plaques. ****, p < 0.0001, Student t test; n = 10. (D) Western blots of infected cells at 24 h p.i. including densitometry. A representative Western blot is shown. **, p < 0.01, Student t test; n = 3. Data are representative of 3 experiments. WL = whole lysate.

Fig 4: Silencing TBK1 increases CVB infection.HeLa cells were treated with siRNA targeting TBK1 (siTBK1) or scrambled RNA (siSCRAMBLE) and subsequently infected with eGFP-CVB at MOI 0.01. (A) Fluorescence microscopy of infected HeLas at 24 h postinfection (p.i.). Phase contrast images show similar cell density at 24 h p.i. Scale bars represent 100 µm. (B) Extracellular viral titers of infected cells at 24 h as measured by plaque assay. **, p < 0.01, Student t test; n = 3. (C) Western blots of infected cells at 0 h, 6 h, and 24 h p.i. including densitometry. A representative Western blot is shown. **, p < 0.01, Student t test; n = 8. Data are representative of 4 experiments. WL = whole lysate.

Fig 5: Silencing TBK1 impairs autophagic flux.HeLa cells were treated with siRNA targeting TBK1 (siTBK1) or scrambled RNA (siSCRAMBLE). (A) Western blot of infected cells at 0 h, 6 h, and 24 h p.i. at MOI 0.01. Densitometric quantification of Western blots. *, p < 0.05, ***, p < 0.001, two-way ANOVA; n = 4. (B) Western blot of cells treated with vehicle or bafilomycin (100 nM) for 2 h. Densitometric quantification of Western blots. **, p < 0.01, ***, p < 0.001, two-way ANOVA; n = 3. Data are representative of 3 experiments. WL = whole lysate.