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. 2020 Nov 4;10(1):19068.
doi: 10.1038/s41598-020-76227-7.

Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes

Yasir Mohamud  1   2 , Junyan Shi  1   2 , Hui Tang  1   3 , Pinhao Xiang  1   2 , Yuan Chao Xue  1   2 , Huitao Liu  1   2 , Chen Seng Ng  1   2 , Honglin Luo  4   5
Affiliations

Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes

Yasir Mohamud et al. Sci Rep. .

Abstract

Coxsackievirus B3 (CVB3) is a single-stranded positive RNA virus that usurps cellular machinery, including the evolutionarily anti-viral autophagy pathway, for productive infections. Despite the emergence of double-membraned autophagosome-like vesicles during CVB3 infection, very little is known about the mechanism of autophagy initiation. In this study, we investigated the role of established autophagy factors in the initiation of CVB3-induced autophagy. Using siRNA-mediated gene-silencing and CRISPR-Cas9-based gene-editing in culture cells, we discovered that CVB3 bypasses the ULK1/2 and PI3K complexes to trigger autophagy. Moreover, we found that CVB3-induced LC3 lipidation occurred independent of WIPI2 and the transmembrane protein ATG9 but required components of the late-stage ubiquitin-like ATG conjugation system including ATG5 and ATG16L1. Remarkably, we showed the canonical autophagy factor ULK1 was cleaved through the catalytic activity of the viral proteinase 3C. Mutagenesis experiments identified the cleavage site of ULK1 after Q524, which separates its N-terminal kinase domain from C-terminal substrate binding domain. Finally, we uncovered PI4KIIIβ (a PI4P kinase), but not PI3P or PI5P kinases as requisites for CVB3-induced LC3 lipidation. Taken together, our studies reveal that CVB3 initiates a non-canonical form of autophagy that bypasses ULK1/2 and PI3K signaling pathways to ultimately converge on PI4KIIIβ- and ATG5-ATG12-ATG16L1 machinery.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
CVB3-induced LC3 puncta and lipidation are dependent on ATG5 and ATG16L1. (A) HEK293A were either sham-infected with PBS or infected with CVB3 for indicated time-points. Western blotting was performed to detect LC3 lipidation and ACTB expression (loading control). Levels of LC3-II were quantified by densitometric analysis, normalized to ACTB and presented as fold changes in the right panel (mean ± SD, n = 3, analyzed by one-way ANOVA with Tukey’s post-test). (B) Schematic diagram of canonical autophagy pathway induced by amino acid deprivation. (C) ATG5- or ATG16L1-knockout (KO) HEK293A cells generated via CRISPR-Cas9 editing, or wildtype (WT)-HEK293A cells were either sham- or CVB3-infected for 16 h. Cell lysates were harvested and probed for LC3, ATG5, and ATG16L1 by Western blotting. Densitometric analysis was carried out as above (the first lane is arbitrarily set a value of 1) and the results are presented under each blot. (D) WT- or Atg5−/− MEFs transfected with mRFP–GFP–LC3 construct were sham- or CVB3-infected for 8 h. GFP and RFP signal was captured by confocal microscopy. Mean LC3 puncta per cell was quantified (n ≥ 10 cells) and analyzed by one-way ANOVA with Tukey post-test (right).
Figure 2
Figure 2
CVB3-induced LC3 lipidation occurs independent of FIP200 and ATG13. (A) FIP200-KO HEK293A cells were established through CRISPR-Cas9 gene editing. Knockout efficiency was validated by western blotting (left panel). WT-HEK293A or FIP200-KO cells were cultured in either normal medium, HBSS starvation medium, or starvation medium supplemented with 200 nM bafilomycin (BAF) for 2 h (middle panel). WT or FIP200-KO cells were sham- or CVB3-infected for 16 h (right panel). Western blotting was performed for analysis of LC3 lipidation. (B,C) Schematic of siRNA-based gene silencing and CVB3 infection schedule (left), HEK293A cells were transiently transfected with scrambled siRNA control (siCON) or siRNAs against FIP200 (B) or ATG13 (C) for 48 h. Cells were then subjected to sham or CVB3 infection for an additional 16 h. Western blotting was conducted to determine knockdown efficiency and LC3 lipidation. Densitometry was measured as above and the results are presented either underneath the blots or in the right panel (mean ± SD, n = 3, analyzed by one-way ANOVA with Tukey’s post-test). (D) HEK293A cells were treated with a selective ULK1/2 kinase inhibitor (MRT68921, 5 µM) under starvation for 2 h in the presence or absence of 200 nM BAF (left panel). HEK293A cells were sham- or CVB3-infected for 16 h with or without 5 µM MRT68921 (right panel). Western blotting was performed to examine LC3 levels and the quantified results are shown underneath the blots.
Figure 3
Figure 3
CVB3-induced LC3 lipidation is independent of BECN1 and PIK3C3. (A) BECN1-KO HEK293A cells were established through CRISPR-cas9 editing. Knockout efficiency was verified by western blotting (left panel). WT or BECN1-KO cells were cultured in either normal medium, HBSS starvation medium, or starvation medium supplemented with 200 nM BAF for 2 h, followed by western blot analysis of LC3 (middle panel). WT or BECN1-KO cells were sham- or CVB3-infected for 16 h, followed by western blot assessment of LC3 (right panel). (B,C) BECN1 (B) and PIK3C3 (C) were transiently silenced in HEK293A cells via siRNA treatment for 48 h. Cells were then subjected to sham or CVB3 infection for 16 h. Cells were harvested and subjected to western blot analysis of LC3, BECN1, and PIK3C3. Densitometric results are presented either underneath the blots or in the right panel (mean ± SD, n = 3, analyzed by one-way ANOVA with Tukey’s post-test).
Figure 4
Figure 4
CVB3-induced LC3 lipidation is independent of ATG9 and WIPI2. (A,B) WIPI2 (A) and ATG9A (B) were transiently silenced in HEK293 cells through siRNA treatment for 48 h. Cells were then sham- or CVB3-infected for 16 h. Western blotting was conducted to verify the knockdown efficiency and to examine LC3 levels. Densitometric results are presented either underneath the blots or in the right panel (mean ± SD, n = 3, analyzed by one-way ANOVA with Tukey’s post-test). (C) WIPI2-KO and ATG9A-KO HEK293A cells, generated through CRISPR-Cas9 gene editing, were sham- or CVB3-infected as above, followed by western blot analysis of LC3, WIPI2, and ATG9A. Densitometry was measured and presented as above.
Figure 5
Figure 5
CVB3 targets several autophagy proteins in HEK293A cells. (A–C) HEK293A cells were sham- or CVB3-infected for 8 h, 16 h, or 24 h as indicated. Cell lysates were harvested and probed for ULK1, ATG13, FIP200, and ULK2, components of the ULK1/2 complex (A), BECN1, UVRAG, PIK3C3, and ATG14, components of the PI3K complex (B), and WIPI2 and ATG9A, components of the WIPI complex (C). Protein levels were quantified and presented underneath each western blot as above.
Figure 6
Figure 6
Viral proteinase 3C cleaves ULK1 after glutamine 524. (A) In vitro cleavage assay was performed by incubating lysates from HeLa cells with vehicle (−), purified wildtype 3C (3Cwt), or catalytically inactive 3C (C147A) mutant (3Cmut) proteins, followed by western blot analysis of ULK1 using an antibody that recognizes an internal region (amino acids 511–750) of ULK1. Arrow denotes the cleavage fragment. (B) HeLa cells were transfected with 3 × Flag-ULK1 together with either empty vector, myc-3Cwt, or myc-3Cmut as indicated. After 24 h, cell lysates were collected and analyzed by western blotting with antibodies against FLAG. (C) HeLa cells were infected with CVB3 for 7 h in the presence or absence of a pan-caspase inhibitor z-VAD-FMK (zVAD, 50 µM) or DMSO (vehicle). Western blotting was performed with antibodies against ULK1. Activation of caspase-3 was examined using an anti-cleaved caspase-3 antibody. Densitometry was measured as above. (D) HeLa cells were co-transfected with 3 × FLAG-ULK1WT or 3 × FLAG-ULK1Q524L (Glutamine 524 mutated to Leucine), together with empty vector, myc-3Cwt, or myc-3Cmut. Western blotting was performed with an anti-FLAG antibody. (E) Schematic illustration of the structural domains, the identified cleavage site, the antibody recognition regions, and the resulting cleavage products of ULK1. GIR, GABARAP interacting region; CTD, C-terminal domain. (F) HEK293 cells were transfected with either vector control, WT-ULK1 or non-cleavable ULK1 (ULK1-Q524L) for 24 h followed by CVB3 infection for an additional 8 h. Supernatant was collected for viral titer measurement (mean ± SD, n = 3, analyzed by unpaired Student t-test).
Figure 7
Figure 7
PI4KIIIβ is involved in CVB3-induced LC3 lipidation. (A) Schematic diagram of the proposed role of PI4KIIIβ in autophagy and the known function in enterovirus replication. (B) PI4KIIIβ was transiently silenced in HEK293A cells using siRNA for 48 h. Cells were then subjected to sham or CVB3 infection for 16 h, followed by western blot analysis of LC3 and PI4KIIIβ. Densitometry was measured as above, and the results are presented underneath and in right panel (mean ± SD, n = 3. Analyzed by one way ANOVA with Tukey’s post-test). (C) HEK293A cells, transfected with control or PIK3C3, PI4KIIIβ, or PIKfyve siRNAs for 48 h, were sham- or CVB3-infected. Western blotting was performed for detection of LC3, VP1, PIK3C3, PI4KIIIβ, or PIKfyve, and quantified as above (right panel) (mean ± SD, n = 3. Analyzed by one way ANOVA with Tukey’s post-test). N.S. not significant.
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