Fig 1.
The impact of pharmacological autophagy modulators and key autophagy-related genes on poxvirus replication.
Human A549 (A) or DF-1 cells (B) were infected in triplicate with VACV-WR or MVA at 3 PFU/cell in the presence of rapa. or baf. A1 at the indicated concentrations. Virus yield at 24 hpi was determined on BS-C-1 (VACV-WR) or DF-1 (MVA) cells. (C) A549 cells were infected with 3 PFU/cell VACV-WR for 2 h, and then cells were treated with baf. A1 (0.1μM) or not, and samples were collected at 24 hpi. The proteins from the samples were extracted and were enzymatically digested, and then the VACV proteins were quantified by LC-MS/MS, followed by proteomic analysis. Flowchart was created with BioGDP.com. (D) Heatmap analysis of early viral proteins of VACV-WR. (E) Heatmap analysis of intermediate-late proteins of VACV-WR. (F) Strategy of the time-of-drug-addition assay. All samples were infected with the VACV-WR between 0 h and 2 h, and the course of baf. A1 addition was set into seven intervals (I–VII). (G) Viral titers of VACV-WR were quantified via plaque assay. (H) A549 cells were infected with 3 PFU/cell VACV-WR or MVA for 8 h, followed by the addition of baf. A1 at indicated concentrations. Virus yield at 24 hpi was determined on BS-C-1 (VACV-WR) or DF-1 (MVA) cells. (I-J) A549 cells were pretreated with DMSO or 3-MA (10, 50, 100 μM) for 2 h and then were infected with 3 PFU/cell VACV-WR or MVA for 24 h. Viral titers of VACV-WR or MVA were quantified on BS-C-1 or DF-1 cells. (K-N) A549 cells were transfected with si-NC as a negative control, siATG3 or siATG7 for 48 h and were subsequently infected with VACV-WR (K-L) or MVA (M-N) at 3 PFU/cell for 24 h. Viral titers of VACV-WR or MVA were quantified on BS-C-1 or DF-1 cells. (O-P) A549 cells were transfected with si-NC as a negative control, siATG16L1 or siBECN1 for 48 h and were subsequently infected with VACV-WR at 3 PFU/cell for24 h, and virus titers were determined by a plaque assay on BS-C-1 cells. (Q) WT A549 cells (NC), A549 cells stably expressing shATG16L1 (ATG16L1-KD) and A549 cells stably expressing shATG16L1 cells transfected with KD-resistant ATG16L1 (ATG16L1-KD + ATG16L1) were infected with VACV-WR at 3 PFU/cell for 24 h.Virus titers were determined by a plaque assay on BS-C-1 cells. Data in Fig 1A, 1B, and 1G-Q represent the mean values ± SD of three independent biological experiments (N = 3 biological replicates). P-values were calculated using the one-way ANOVA. Statistics: n.s., not significant, p > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 2.
VACV infection promoted activation of autophagy.
A549 were infected with MVA (A) or VACV-WR (B) at 3 PFU/cell in the presence or absence of rapa. (100nM) or baf. A1 (0.1μM), and cell lysates were collected at indicated time points and subjected to Western blotting analysis with anti-SQSTM1, anti-MAP1LC3B, anti-I3, and anti-GAPDH antibodies. The quantification of MAP1LC3B-II and SQSTM1 was shown in Fig 2C-F. (G) A549 cells stably expressing GFP-MAP1LC3B on coverslips were infected with VACV-WR, MVA at 3 PFU/cell or treated with 3-MA (1mM) or rapa. (100nM) and cells were fixed at 12 hpi, stained with Hoechst and observed with a fluorescent confocal microscope. (H) Numbers of GFP-MAP1LC3B puncta (representing autophagosomes) were quantitated with Image J. Three independent biological replicates were performed, with 15 cells quantified per replicate. Scale bars represent 10 μm. Data in Fig 2A, 2B, and 2G are representative of three independent experiments (N = 3 biological replicates). Statistics: n.s., not significant, p > 0.05; **P < 0.01; ****P < 0.0001 by one-way ANOVA.
Fig 3.
VACV-WR and MVA differed in their ability to inhibit autolysosome formation.
(A) Diagram illustrating the working principle of the reporter plasmid mCherry-GFP-MAP1LC3B. (B) A549 cells grown on coverslips were transfected with the mCherry-GFP-MAP1LC3B plasmid for 24 h and then were mock-infected, infected with VACV-WR or MVA at 3 PFU/cell at the presence or absence of baf. A1(50nM), or treated with CQ (40μM) or rapa. (100nM) for 12 h. Cells were fixed, stained with Hoechst and analyzed with a fluorescent confocal microscope. Scale bars indicate 10 μm. (C) The graph shows the quantification of autophagosomes. Three independent biological replicates were performed, with 15 cells quantified per replicate. And bars represent mean values ± SD of three independent biological replicates. (D) A549 cells stably expressing MAP1LC3B were infected with VACV-WR, MVA at 3 PFU/cell or treated with baf. A1 or rapa. as described above. Lysotracker was added to live cells to stain lysosomes. (E) Images were taken as described above and quantification of colocalization was analyzed using Image J software. Three independent biological replicates were performed, with 15 cells quantified per replicate. Data in Fig 3C and 3E represent the mean values ± SD of three independent biological replicates (N = 3 biological replicates). Statistics: n.s., not significant, p > 0.05; ****P < 0.0001 by two-sided Student’s t test or one-way ANOVA.
Fig 4.
The effect of A52 on the fusion of autophagosome with lysosome.
(A) A549 cells stably expressing GFP-MAP1LC3B were left untreated, infected with WR or MVA at 3 PFU/cell in the presence or absence of rapa. (100nM) or transiently transfected with a plasmid encoding a Flag-tagged A52 (1500 ng) prior to MVA infection. At 12 hpi, cells were then fixed, permeabilized, blocked, and stained with primary antibodies to LAMP1 and followed by fluorescent conjugated secondary antibodies. Hoechst was used to stain nucleus. Scale bars represent 10 μm. Numbers of autolysosomes were quantitated in Fig 4B. Three independent biological replicates were performed, with 15 cells quantified per replicate. (C) Human A549 cells were transfected with a vector encoding A52-Flag at concentrations of 0.1, 0.5, and 1.5 μg/mL for 24 h and then infected with MVA at 3 PFU/cell for 12 h. WR-infected cells and cells treated with baf. A1 and rapa. were included as controls. Cell lysates were analyzed using SDS-PAGE followed by Western blotting analysis with anti-SQSTM1, anti-Flag, anti-MAP1LC3B, or anti-GAPDH antibodies. The quantification of MAP1LC3B-II and SQSTM1 was shown in Fig 4D. (E) Human A549 cells were infected with WR, WR-ΔA52, vA52-rev, MVA or MVA + A52 at 3 PFU/cell or treated with baf. A1 (0.1μM) or rapa. (100nM) for 12 h. Cell lysates were analyzed using SDS-PAGE and Western blotting with anti-SQSTM1, anti-I3, anti-MAP1LC3B, or anti-GAPDH antibodies. The quantification of MAP1LC3B-II and SQSTM1 was shown in Fig 4F. (G) A549 cells were infected with WR, WR-ΔA52, MVA, MVA + A52 at 3 PFU/cell or treated with baf. A1 (0.1μM) or rapa. (100nM) for 12 h. Cells were then fixed, permeabilized, blocked, and stained with primary antibodies to LAMP1 or MAP1LC3B and followed by fluorescent conjugated secondary antibodies. Hoechst was used to stain nucleus. Scale bars represent 10 μm. Numbers of autolysosomes were quantitated in Fig 4H. Three independent biological replicates were performed, with 15 cells quantified per replicate. Data in Fig 4D and 4F are shown as dots, and the bar represents the mean value. Data in Fig 4B and 4H represent the mean values ± SD of three independent experiments (N = 3 biological replicates). Data in Fig 4A-H are representative of three independent experiments. Statistics: n.s., not significant, p > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA.
Fig 5.
A52 is associated with human SNAP29.
Human A549 cells were co-transfected with plasmids encoding Myc-tagged SNAP29 and Flag-tagged A52 for 36 h. Cell lysates were pre-cleared with control magnetic beads and then incubated with Flag-conjugated (A) or Myc-conjugated (B) beads at 4˚C for 18 h. Beads were extensively washed, and proteins were eluted with SDS loading buffer and analyzed using SDS-PAGE and Western blotting described above. (C) A549 cells were mock-infected or infected with MVA + A52 at 3 PFU/cell for 24 h. Cell lysates were pre-cleared with control magnetic beads or Flag-conjugated beads at 4˚C for 18 h. Beads were extensively washed, and proteins were eluted with SDS loading buffer and analyzed using SDS-PAGE and Western blotting with anti-SNAP29, anti-Flag antibodies. (D) A549 cells grown on coverslips were infected with MVA or MVA + A52 at 3 PFU/cell for 12h. Cells were then fixed, permeabilized, blocked, and stained with primary antibodies to SNAP29 or Flag and followed by fluorescent conjugated secondary antibodies. Hoechst was used to stain nucleus. The panels below show the fluorescence intensity profile of SNAP29 (green) and A52 (red) measured along the line drawn by Image J. Scale bars represent 10 μm. (E) A series of SNAP29 truncation mutants were constructed and illustrated. (F) A549 cells were co-transfected with vectors encoding Myc-tagged SNAP29 or its mutants, and a Flag-tagged A52 for 36 h. Cell lysates were pre-cleared with control magnetic beads or Flag-conjugated beads at 4˚C for 18 h followed by extensive washing. Proteins were eluted with SDS loading buffer and resolved by SDS-PAGE followed by Western blotting analysis using primary antibodies for Flag, Myc and GAPDH. Data in Fig 5A, 5B, 5C, 5D, and 5F are representative of three independent experiments. (G) A549 cells grown on coverslips were transfected with vectors encoding Myc-tagged SNAP29 or its mutants, and a Flag-tagged A52 for 24 h. Cells were then fixed, permeabilized, blocked, and stained with primary antibodies to Myc or Flag and followed by fluorescent conjugated secondary antibodies. Hoechst was used to stain nucleus. The right panels show the fluorescence intensity profile of A52-Flag (green) and Myc-tagged SNAP29 or its mutants (red) measured along the line drawn by Image J. Scale bars represent 10 μm. Data in Fig 5A, 5B, 5C, 5D, 5F and 5G are representative of three independent experiments (N = 3 biological replicates).
Fig 6.
A52 hampered the interaction between SNAP29 and its binding partners STX17 and VAMP8.
Human A549 cells were co-transfected with plasmids encoding Myc-tagged SNAP29, HA-tagged STX17, and Flag-tagged A52 for 36 h. Cell lysates were pre-cleared with control magnetic beads and then with Myc- (A) or HA-conjugated (B) beads at 4˚C for 18 h. Beads were extensively washed and proteins were eluted with SDS loading buffer and resolved by SDS-PAGE and Western blotting analysis. (C) A549 cells on coverslips were co-transfected with SNAP29-Myc, STX17-HA and A52-Flag for 24 h. Cells were then fixed, stained with anti-Myc, anti-HA antibodies and Hoechst and images were taken with a fluorescent confocal microscope. The right panels show the fluorescence intensity profile of STX17-HA (green) and Myc-tagged SNAP29 (red) measured along the line drawn by Image J. Scale bars represent 10 μm. (D) A549 cells were co-transfected with SNAP29-Myc and STX17-HA for 24 h, and then infected with WR, WR-ΔA52, and vA52-rev for 12 h. Cell lysates were precleared with control magnetic beads and then incubated with Myc-conjugated beads at 4˚C for 18 h. Beads were extensively washed and proteins were eluted with SDS loading buffer and resolved by SDS-PAGE and Western blotting analysis. (E) A549 cells were co-transfected with SNAP29-Myc, VAMP8-HA, and A52-Flag for 36 h. Cell lysates were processed as described in (D). (F) A549 cells were co-transfected with SNAP29-Myc and VAMP8-HA for 24 h, and then infected with WR, WR-ΔA52, and vA52-rev for 12 h. Cell lysates were processed as described in (D). (G) Schematic Diagram of the FRET (Fluorescence Resonance Energy Transfer). (H) A549 cells were transfected with GFP-STX17 (donor) and mCherry-SNAP29 (Acceptor) for 24 h, and then were infected with 3 pfu/cell of MVA or MVA + A52 virus for 12 h. FRET-FLIM analysis was performed to assess changes in the donor fluorescence lifetime. Cells co-transfected with GFP-STX17 and mCherry-SNAP29 served as the FRET positive control, while cells transfected with GFP-STX17 alone served as the negative control. The last column displays the fluorescence lifetime of the donor GFP-STX17, pseudo-color coded from low (dark blue) to high (yellow/red). (I) A549 cells were transfected with GFP-VAMP8 (donor) and mCherry-SNAP29 (Acceptor) for 24 h, and then were infected with 3 pfu/cell of MVA or MVA + A52 virus for 12 h. FRET-FLIM analysis was performed to assess changes in the donor fluorescence lifetime. Cells co-transfected with GFP-VAMP8 and mCherry-SNAP29 served as the FRET positive control, while cells transfected with GFP-VAMP8 alone served as the negative control. The last column displays the fluorescence lifetime of the donor GFP-VAMP8, pseudo-color coded from low (dark blue) to high (yellow/red). (J) Box plots showing median GFP lifetimes in different conditions within the plate: red: only donor (GFP-STX17-GFP); blue: GFP-STX17 (donor) + mCherry-SNAP29 (Acceptor); green: GFP-STX17 (donor) + mCherry-SNAP29 (Acceptor)+MVA + A52; orange: GFP-STX17 (donor) + mCherry-SNAP29 (Acceptor)+MVA. (K) Box plots showing median GFP lifetimes in different conditions within the plate: red: only donor (GFP-VAMP8); blue: GFP-VAMP8 (donor) + mCherry-SNAP29 (Acceptor); green: GFP-VAMP8 (donor) + mCherry-SNAP29 (Acceptor)+MVA + A52; orange: GFP-VAMP8 (donor) + mCherry-SNAP29 (Acceptor)+MVA. Data in Fig 6A-F, 6H-I are representative of three independent experiments (N = 3 biological replicates). Statistics: n.s., not significant, ****P < 0.0001 by one-way ANOVA.
Fig 7.
VACV-A52 facilitated proteasome-mediated SNAP29 degradation.
A549 cells were transfected with vectors expressing VACV-WR-A52 (A) or LSDV-A52 (B) at concentrations of 0.2, 0.5, and 1.5 μg/mL for 36 h. Cell lysates were resolved by SDS-PAGE followed by Western blotting analysis with anti-SNAP29, anti-Flag, anti-GAPDH primary antibodies. A549 cells were transfected with plasmids encoding A52 from VACV (C) or LSDV (D) at 1.5 μg/mL and total proteins were collected at 0, 24, 36, and 48 h post transfection. Protein samples were resolved with SDS-PAGE and Western blotting analysis with primary antibodies for human SNAP29, Flag or GAPDH. (E) A549 cells were infected with VACV-WR, VACV-WR-ΔA52, MVA or MVA + A52 for 24 h, and cell lysates were resolved by SDS-PAGE followed by Western blotting analysis with anti-SNAP29, anti-I3, anti-GAPDH antibodies. (F) A549 cells were transfected with the A52-Flag or empty vector plasmids for 24 h, and then the cells were treated with Baf. A1 (0.1 μM), CQ (40 μM), MG132 (5 μM) or Z-VAD-FMK (10 μM) for 12 h. DMSO was included as a negative control. Western blotting analysis was conducted to detect endogenous SNAP29 synthesis using protocols described above. (G-H) A549 cells were transfected with A52-Flag for 24 h in the presence or absence of MG132 or Epoxomicin at various concentrations. After 12 h, cell lysates were analyzed using SDS-PAGE and Western blotting analysis using anti-SNAP29, anti-Flag, anti-GAPDH antibodies. (I) A549 cells were infected with MVA or MVA + A52 at 3 PFU/cell. After 2 h, cells were washed with PBS and then transfected with an empty vector or vector encoding HA-tagged Ubiquitin for 36 h. Cell lysates were then precleared with control magnetic beads and then incubated with SNAP29-conjugated beads at 4˚C for 18 h. Beads were extensively washed and proteins were eluted with SDS-loading buffer and resolved by SDS-PAGE followed by Western blotting analysis with primary antibodies for Flag (A52), SNAP29 or HA (ubiquitin). Data in Fig 7A-I are representative of three independent experiments (N = 3 biological replicates).
Fig 8.
SNAP29 inhibits the replication of MVA in A549 cells.
Human A549 cells were transfected with siNC or siSNAP29 (A) for 48 h and then infected in triplicates with MVA or MVA + A52 (B) and WR or WR-ΔA52 (F) at 3 PFU/cell. Viruses were harvested at 24 hpi and virus titers were determined by plaque assay on DF-1 cells or BS-C-1 cells. Human A549 cells were transfected with SNAP29-Myc (C) for 24 h and then infected in triplicates with MVA or MVA + A52 at 0.01 (D) or 3 PFU/cell (E). Viruses were collected at 48 or 24 hpi and titers were determined by plaque assay on DF-1 cells. Human A549 cells were transfected with SNAP29-Myc (G) for 24 h and then infected in triplicates with WR or WR-ΔA52 at 0.01 (H) or 3 PFU/cell (I). Viruses were collected at 48 or 24 hpi and virus titers were determined by plaque assay on BS-C-1 cells. (J-K) A549 cells were transfected with SNAP29-Myc at concentrations of 0.2, 0.5, or 1.5 μg/mL for 24 h and were infected in triplicate with MVA, MVA + A52, VACV-WR or VACV-WR-ΔA52 at 3 PFU/cell for 24 h and virus titers were determined by plaque assay on DF-1 or BS-C-1.(Data in Fig 8B, 8D, 8E, 8F, 8H, 8I, and 8K are shown as dots, and the bar represents the mean value. Data in Fig 8A-K are representative of three independent experiments (N = 3 biological replicates). Statistics: n.s., not significant, p > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by two-sided Student’s t test.
Fig 9.
Mechanism of A52 in modulating the fusion of autophagosome and lysosome.
The figure was created in BioRender. Deng, Y. (2026) https://BioRender.com/e0uikcg.