Fig 2: Analysis of LC3 family protein expression and lipidation. (A) Total cell lysates from primary human fibroblasts treated with BafA1, Ex527 or TSA for 48h were analyzed by Western blot as indicated. (B–E) Densitometric quantifications of the immunoreactive bands from n = 3 independent experiments. Raw numbers were first normalized to tubulin, and the resulting values were related to the similarly tubulin-normalized control (Ctrl). (B) Autophagic flux analyses in cells treated as indicated for 48h. Analysis was performed for each ATG8 protein by determining the difference, between BafA1-treated and BafA1-untreated cells, of the intensity of the lipidated bands running below the unlipidated bands [Flux = (LC3II/Tub)with BafA1—(LC3II/Tub)without BafA1]. Significant changes (by one-way ANOVA on ranks) versus the control: * p ≤ 0.05; ** p ≤ 0.01. (C) Raw intensities of all lipidated protein bands (tubulin-normalized). Significant changes (by two-way ANOVA) versus BafA1-untreated cells: * p ≤ 0.05; ** p ≤ 0.01. (D) Raw intensities of all lipidated and unlipidated protein bands (tubulin-normalized). Statistical analysis was done as in (C). (E) Quantification of SIRT1 and p62 expression (tubulin-normalized).

Fig 3: 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 4: Analysis of EGF-induced EGFR phosphorylation, activation of downstream signaling and gene expression in GABARAP SKO and Huh7.5 control cells. (A) Huh7.5 and GABARAP SKO cells were treated with 40 ng/mL EGF for the indicated times. Afterwards, activating phosphorylations of the EGFR (B) and downstream PI3K/AKT (C) and MAPK/ERK (D) signaling pathways were analyzed by immunoblot. Quantification of phosphorylated proteins was performed by normalization to the corresponding total protein levels and calculated as percentage of Huh7.5 control cells after 10 min. of EGF treatment (t = 10). (E + F) Huh7.5 and GABARAP SKO cells were treated with the indicated concentrations of EGF for 180 min., followed by cell lysis, RNA extraction, reverse transcription and quantitative real-time PCR (RT-PCR). Expression of target genes CXCL8 (E) and CXCL1 (F) was normalized to succinate dehydrogenase (SDHA) as reference gene and is expressed relative to unstimulated control cells. (G) Protein expression of GABARAP paralogs GABARAPL1 and GABARAPL2 was analyzed in Huh7.5 and GABARAP SKO cells and densitometric analysis performed to determine GABARAPL1 and GABARAPL2 protein levels in GABARAP SKO cells compared to Huh7.5 controls (H). Representative blots are shown for a summary of n = 3 independent experiments. Error bars represent standard errors of means. Asterisks mark significant differences versus the corresponding time point or concentration of control cells as calculated using independent t-test. p = 0.05 = *. (E,F,H) individual experiments are color-coded. Figure S6C shows uncropped source blots.

Fig 5: Silencing GABARAPL1 and GABARAPL2 reduces CVB infection and secretion into EVs.(A) HeLa cells were treated with siRNA targeting GABARAPL1 and GABARAPL2 (siGABA1/2) and subsequently infected with eGFP-CVB for 24 h. Fluorescence microscopy of HeLa cells infected at 24 h p.i. at MOI 0.01. Phase contrast images show similar cell density at 24 h p.i. Scale bars represent 100 μm. (B) Extracellular viral titers of free virus (free) or EV virus (EV) at 24 h p.i. at MOI 0.1 as measured by plaque assay. ***, p < 0.001, ****, p < 0.0001, two-way ANOVA; n = 3. (C) Western blots of infected cells at 24 h p.i. at MOI 0.01. Densitometric quantification of Western blots. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, two-way ANOVA; n = 3. (D) siSCRAMBLE or siGABA1/2 cells were treated with vehicle or bafilomycin (100 nM) for 2 h. Cell lysates were analyzed by Western blot and densitometry quantification was performed. *, p < 0.05, two-way ANOVA; n = 3. Data are representative of 3 experiments. WL = whole lysate.