Fig 1.
NDV infection induces LMP in HeLa cells.
(A&B) HeLa cells were infected with Herts/33 at 0.01 MOI for the indicated time points. Torin 1 (1 μM, 4h) and Baf A1 (1 μM, 4h) were used as positive and negative controls, respectively. The fluorescence signals were detected by confocal microscopy (A) and flow cytometry (B) after staining the cells with Lysotracker Red (100 nM) for 1h. Scale bars, 50 μm. (C) HeLa cells were infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. Then the AO red and green fluorescence signals were detected by confocal microscopy following incubation with AO (1 μg/mL) for 30 min. Scale bars, 20 μm. (D&E) Quantitation of AO red (D) and green (E) fluorescence signals by ImageJ software. (F) HeLa cells were infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. LLoMe (1 mM, 4h) was used as a positive control. Then the cells were stained with rabbit anti-galectin 3 (green) and mouse anti-LAMP1 (red) antibodies and observed by confocal microscopy. Scale bars, 20 μm. (G) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 were measured by ImageJ software. Error bars represent SDs for triplicate analyses of >100 cells/sample (D&E), or SDs for 15 cells (G). All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 2.
NDV infection induces the leakage of CTSB and CTSD from the lysosomal lumen to the cytoplasm in HeLa cells.
(A) HeLa cells infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. Indicated proteins in both lysosomal and cytoplasmic fractions extracted from the cells were detected by Western blotting. Immature and mature types of cathepsins were indicated as “IM” and “M”, respectively. (B-E) The relative intensity of lysosomal CTSB (B) and lysosomal CTSD (D) or cytoplasmic CTSB (C) and cytoplasmic CTSD (E) were quantified by ImageJ software. LAMP3 and α-Tubulin were used as normalized controls for lysosomal and cytoplasmic fractions, respectively. (F&G) HeLa cells were infected with Herts/33 at 0.01 MOI for the indicated time points or mock-infected. The activity of cytoplasmic CTSB (F) and CTSD (G) was measured as described in Materials and Methods. The obtained fluorescence intensity was normalized to the concentration of individual protein. (H&K) HeLa cells were infected with Herts/33 at 0.01 MOI for indicated timepoints or mock-infected. The colocalizations of CTSB (H) or CTSD (K) with LAMP3 were observed using confocal microscopy after immunostaining the cells with indicated antibodies. Scale bars, 20 μm. (I&J) Manders’ Colocalization Coefficients of CTSB (I) or CTSD (J) with LAMP3 were quantified by ImageJ software. Error bars represent SDs for triplicate analyses of three independent experiments (B-F) or SDs for 15 cells (I&J). All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 3.
LMP promotes NDV-induced apoptosis through the involvement of CTSB and CTSD.
(A) Schematic diagram of the experimental design for (B-G). HeLa cells or DF-1 cells were infected with Herts/33 at 0.01 MOI for 24h. Then, the cells were treated with Ca-074 (20 μM), Pep A (20 μM), DMSO, or mock-treated for 6h. (B&C&E&F) The apoptosis level of HeLa cells (B) or DF-1 cells (E) was measured by AnnexinV-FITC/PI staining using flow cytometry, and quantitation of the apoptosis rate of HeLa cells (C) and DF-1 cells (F) are shown, respectively. (D&G) The NP protein in HeLa cells (D) or DF-1 cells (G) was detected by Western blotting. (H) Schematic diagram of the experimental design for (I-M). HeLa cells were transfected with si-CTSB, si-CTSD, si-NC, or mock-transfected for 48h. Then, the cells were infected with Herts/33 at 0.01 MOI or mock-infected for 12h. (I-M) The indicated proteins were detected by Western blotting (I). The relative intensity of cleaved caspase 3 (J), cleaved caspase 7 (K), cleaved caspase 9 (L), and cleaved PARP1 (M) in NDV-infected cells were quantified by ImageJ software. GAPDH was used as a normalized control. All error bars represent SDs for triplicate analyses of three independent experiments. All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 4.
LMP promotes NDV-induced apoptosis by inducing mitochondrial dysfunction via CTSB and CTSD.
(A&B) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. The MMP level was detected by flow cytometry (A) or fluorescence microscope (B) using JC-1 probe staining following infection with Herts/33 at 0.01 MOI for 12h or mock-infected. Scale bars, 100 μm. (C&D) Quantitation of JC-1 red to green fluorescence ratio detected by flow cytometry (C) and fluorescence microscope (D) by ImageJ software are shown, respectively. (E-G) Indicated proteins were detected by Western blotting in HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints (E). The relative intensity of Bcl-2 (F) and tBid (G) were quantified by ImageJ software. GAPDH was used as a normalized control. (H-J) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. Subsequently, the indicated proteins were detected by Western blotting following infection with Herts/33 at 0.01 MOI or mock-infection for 12h (H). The relative intensity of Bcl-2 (I) and tBid (J) in NDV-infected cells were quantified by ImageJ software. GAPDH was utilized as a normalized control. (K-O) HeLa cells were transfected with si-CTSB or si-CTSD, or si-NC for 48h or mock-transfected. The expression levels of Bax and Cyt C in the cytoplasmic and mitochondrial fractions were determined by Western blotting after infection with Herts/33 at 0.01 MOI or mock-infection for 12h (K). The relative intensity ratio of Bax/GAPDH (L), Cyc/GAPDH (M), Bax/COX IV (N), and Cyc/COX IV (O) were quantified by ImageJ software, respectively. GAPDH was used as a cytoplasmic fraction control and COX IV was used as a mitochondrial fraction control. All error bars represent SDs for triplicate analyses of three independent experiments. All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 5.
CTSB and CTSD exacerbate NDV-induced LMP by inducing the generation of ROS.
(A) Intracellular ROS levels were measured by flow cytometry using DCFH-DA staining in HeLa cells infected with Herts/33 at 0.01 MOI for different timepoints or mock-infected. (B) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h, or mock-transfected, and intracellular ROS levels were measured by flow cytometry using DCFH-DA staining following infection with Herts/33 at 0.01 MOI or mock-infection for 12h. (C) HeLa cells were pretreated with LLoMe (500 nM), DMSO, or mock-untreated for 3h, respectively. Subsequently, the intracellular ROS levels were measured by flow cytometry using DCFH-DA staining cells following infection with Herts/33 at 0.01 MOI or mock-infection for 24h. (D) HeLa cells were treated with NAC (2 mM) for 24h after absorption with Herts/33 at 0.01 MOI for 1h. LMP levels were evaluated by confocal microscopy using indicated antibodies. Scale bars, 20 μm. Manders’ Colocalization Coefficients of galectin 3 with LAMP1 were quantified by ImageJ software and shown on the right. Error bars represent SDs for triplicate analyses of three independent experiments (A-C) or SDs for 15 cells (D). All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 6.
NDV infection induces deglycosylation and degradation of LAMP1 and LAMP2.
(A&B) HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints. The mRNA levels (A) and fluorescence images (B) of LAMP1 and LAMP2 were measured by RT-qPCR or observed by confocal microscopy, respectively. The mRNA levels of LAMP1 and LAMP2 were normalized to GAPDH using the 2−ΔΔCt method. Scale bars, 20 μm. (C-E) HeLa cells infected with Herts/33 at 0.01 MOI for indicated time points or infected with Herts/33 at indicated MOIs for 24h. The protein levels of LAMP1 and LAMP2 (C) or LAMP3, LIMPII, and LAPTM5 (E) were assessed by Western blotting. The deglycosylated LAMP1 and LAMP2 in PNGase F-treated cell lysates were analyzed by Western blotting (E). (F-H) HeLa cells were infected with Herts/33 at 0.01 MOI for 12h or mock-infected. The remaining amounts of LAMP1 and LAMP2 after treatment with CHX (20 μg/mL) for indicated time points were measured using Western blotting (F). The remaining amount of LAMP1 (G) and LAMP2 (H) was calculated as the fold change of the protein present at 0h. (I-K) NDV-infected (0.01 MOI, 6h) HeLa cells were co-treated with CHX (20 μg/mL) and NH4CL (50 mM), or Ca-074 (10 μM), or Pep A (10 μM), or 3-MA (5mM), or MG132 (5 μM) or mock-treated for 12h. LAMP1 and LAMP2 protein levels in cell lysates were analyzed by Western blotting (I). The remaining amounts of LAMP1 (J) and LAMP2 (K) were calculated as the fold change of the protein to mock-treated cells at 0h. All quantitative data represent means ± SD (n = 3). Significance was assessed using Two-way ANOVA with Dunnett’s multiple comparisons test (A), or unpaired two-tailed Student’s t-test (G&H), or One-way ANOVA with Dunnett’s multiple comparisons test (J&K).
Fig 7.
The viral HN protein contributes to NDV-induced LMP and apoptosis.
(A) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with plasmids expressing indicated viral proteins for 48h. (B&C) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with indicated doses of plasmids expressing HN (B) or F (C) protein for 48h. (D) Confocal microscopy images of LMP level in HeLa cells using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies after transfection with indicated plasmids for 48h. Scale bars, 20 μm. (E&F) The interaction between HN and LAMP1 (E) or LAMP2 (F) was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids into HeLa cells for 48h. (G&H) Confocal microscopy images of the interaction between HN and LAMP1 (G) or LAMP2 (H) using anti-HN, anti-LAMP1, or anti-LAMP2 antibodies after infection of HeLa cells with Herts/33 at 0.01 MOI or mock-infected for 24h. Scale bars, 20 μm. (K) HeLa cells were transfected with siRNAs targeting LAMP1 or LAMP2 or plasmids expressing LAMP1 or LAMP2 for 48h. Then LAMP1 or LAMP2 protein levels were detected by Western blotting. The red boxes indicate the siRNA selected for this study. (I&J) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (M) or siRNAs targeting LAMP1 or LAMP2 (N) for 48h. Then the LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with plasmid expressing HN protein for another 48h. Scale bars, 20 μm. (L&M) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (L) for (I) and (M) for (J). (N&O) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (N) or siRNAs targeting LAMP1 or LAMP2 (O) for 48h. Then the apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with plasmid expressing HN protein for 48h. Error bars are SDs for 15 cells (L&M), or SDs for a triplicate analysis of three independent experiments (N&O). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.
Fig 8.
The sialidase activity of the HN protein mediates the deglycosylation and degradation of LAMP1 and LAMP2.
(A) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with plasmid expressing HN protein and treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) at the indicated time points post-transfection. (B&C) HeLa cells were transfected with plasmids expressing HN protein or mock-transfected. Then the cells were treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) or DMSO or untreated for 48h. The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies. Scale bars, 20 μm (B). Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (C). (G) Sialidase activity was measured in HeLa cells transfected with the indicated plasmids for 48h and is presented as relative change to the cells transfected with plasmids expressing wild-type HN. The red dotted line indicates the difference threshold. (F) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with the indicated plasmids or mock-transfected for 48h. (D&E) The interaction between HN and LAMP1 (D) or LAMP2 (E) in HeLa cells was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids for 48h. (H) The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with the indicated plasmids or mock-transfected for 48h. Scale bars, 20 μm. (I) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are calculated by ImageJ software. (J) The apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with indicated plasmids or mock-transfection for 48h. Error bars are SDs for a triplicate analysis of three independent experiments (G&J), or SDs for 15 cells (C&H). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.
Fig 9.
A mechanism model showing the HN protein of NDV triggers LMP and promotes apoptosis.
NDV enters host cells and utilizes its genome for transcription and translation of the HN protein. The HN protein digests the sialic acid at the end of the glycan chains of LAMP1 and LAMP2 via its sialidase activity, leading to desialylation. The desialylated LAMP1 and LAMP2 undergo deglycosylation and degradation in the lysosome by CTSB, leading to LMP and the leakage of CTSB and CTSD. Consequently, these translocated CTSB and CTSD promote the degradation of Bcl-2, the cleavage of Bid into tBid, and the mitochondrial translocation of Bax, thereby inducing MOMP. This process further exacerbates LMP by inducing ROS release while simultaneously activating mitochondria-dependent apoptosis through the release of Cyt C.