Fig 2: 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.

Fig 3: 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 4: In silico sequence and binding partner analysis for the ATG8 protein family. (A) Phylogenetic tree of the ATG8 gene family featuring H. sapiens LC3A, LC3B, LC3B2, LC3C, GABARAP, GABARAP-L1, and GABARAP-L2, D. melanogaster ATG8A and ATG8B as well as S. cerevisiae ATG8. (B) Protein sequence alignment indicating identical (*), conserved (:) and semi-conserved (.) amino acids. Residues that have been found essential for ATG7 binding are indicated by a purple arrow, the conserved lipidation site glycine is marked with blue. (C) Meta-analysis of the amino acid composition of all putative (4-amino-acid) LIR motifs of the indicated number of recognized, exclusive ATG8 interactors as published [18]. The relative amino acid abundance for each position of a LIR motif was calculated and is shown by the indicated color-coding.

Fig 5: vIRF-1 binds directly to GABARAPL1 in an LIR-independent manner.(A) Schematic structure of vIRF-1:PD, Proline-rich domain; DBD, DNA-binding domain; and IAD, IRF-association domain. LC3-interacting regions (LIRs) were searched for using a position-specific scoring matrix (PSSM) via the web-based software iLIR, and a conserved LIR of vIRF-1 with the highest PSSM score was predicted at the N-terminal junction of the IAD, as noted. (B) GST pull-down assay to assess in vitro binding between vIRF-1 and ATG8 proteins (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2). Purified recombinant T7-tagged vIRF-1 (vIRF-1-T7) was pulled down with 1μg of GST-fused ATG8 proteins immobilized on glutathione beads. GST, GST-SUMO, and GST-Ub were used as controls. (C) Co-immunoprecipitation (co-IP) assays for assessment of the intracellular interactions between vIRF-1 and ATG8 proteins in virus-infected cells. iBCBL-1 cells were reactivated by treatment with 1 μg/ml doxycycline (Dox) for 2 days and fractionated into the nuclear and cytoplasmic fractions. The fractions were immunoprecipitated with normal rabbit IgG (nIgG) and rabbit anti-vIRF-1 antibody, and the immunoprecipitated complex was analyzed by immunoblotting with the indicated antibodies. Lamin B1 and lactate dehydrogenase (LDH) were used as nuclear and cytoplasmic fraction markers, respectively. (D) Immunofluorescence assay of the co-localization of vIRF-1 and GABARAPL1 in mitochondria (TOM20) in lytically virus-infected cells. iBCBL-1 cells were reactivated by treatment with Dox for 2 days and were fixed (Fix) with 4% paraformaldehyde before permeabilization (Per) with 0.5% Triton X-100 or after permeabilization with 25 μg/ml of saponin. A rat anti-vIRF-1 antibody was used. Scale bar, 10 μm. (E) Mutation of the core residues in the predicted LIR of vIRF-1: both tryptophan at position 255 (W255) and leucine at position 258 (L258) were replaced with alanine. (F-G) GST pull-down assays for assessment of in vitro interactions of wild-type (WT) and A255/A258 vIRF-1 with GST-LC3B and GST-GABARAPL1. GST alone was used as a control. The whole-cell lysates of 293T cells expressing Flag-tagged vIRF-1 (vIRF-1-Flag) were used as input for the pull-down assay. (G) The intensities of co-precipitated vIRF-1 proteins relative to input were determined, and the data represent the mean ± SD of three independent experiments. ‘ns’, not significant. (H) Co-IP assay. 293T cells were co-transfected with vIRF-1(WT and A255/A258)-Flag with V5-LC3B or V5-GABARAPL1 and immunoprecipitated with anti-Flag antibody.