Figures and data
RVFV infection leads to mitochondrial elongation.
(A) Mitochondria were visualized by immunofluorescence using confocal microscopy. THP-1PMA, Huh7 or A549 cells were infected or mock infected with RVFV for 24 h at MOI = 5. Mitochondria were labeled with anti-Tom20 antibody (red), RVFV were labeled with anti-RVFV-NSs antibody (green) and Nuclei were stained with DAPI (blue). Scale bars, 5 μm (THP-1PMA and Huh7 cells) or 7.5 μm (A549 cells).(B) Sections of mock infected or RVFV-infected THP-1PMA cells (MOI = 1) were analyzed by TEM. Scale bars, 1 μm. Blue dashed squares represent the magnification of regions of interest. Mitochondria are circled in red shown on the right panels. Data are representative of three experiments with similar results.
RVFV infection induces NSs-dependent DRP1 downregulation leading to mitochondrial elongation.
(A) The effects of exogenously expressed RVFV viral proteins on mitochondrial morphology in Huh7 cells was observed by confocal microscopy. Huh7 cells were transfected with empty vector or vectors expressing strep-tagged viral proteins of RVFV for 48 h. Mitochondria were labeled with anti-Tom20 antibody (red), viral proteins of RVFV were labeled with anti-strep antibody (green) and Nuclei were stained with DAPI (blue). Scale bars, 7.5 μm. (B) Huh7 cells were mock infected or infected with the indicated virus for 24 h. Mitochondria were labeled with anti-Tom20 antibody (red), RVFV were labeled with anti-RVFV-NSs antibody (green), RVFVEGFP△NSs were observed through detecting EGFP signal, and Nuclei were stained with DAPI (blue). Scale bars, 7.5 μm. (C) Immunoblot analysis of the proteins that regulate mitochondrial morphology, including MFN1, MFN2, OPA1 and DRP1 in THP-1PMA cells infected with RVFV for the indicated times. (D) Immunoblot analysis of DRP1 protein level in Huh7 cells infected or mock infected with RVFVWT or RVFVEGFP△NSs for 24 h. (E) Immunoblot analysis of DRP1 protein level in Huh7 cells transfected with the plasmids expressing the indicated proteins for 48 h. Data are representative of three experiments with similar results.
DRP1 is cleaved by activated caspase during RVFV infection.
(A-C) Immunoblot analysis of indicated protein levels in Huh7 cells infected or mock infected with RVFV for 2 h, followed by MG132 (5 μM) (A), CQ (10 μM) (B) or Z-VAD-FMK (20 μM) (C) treatment for 22 h. The numbers below each lane represent protein levels relative to Tubulin and expressed relative to the control group (defined as 1) by ImageJ gray value analysis. (D) Immunoblot analysis of DRP1 protein level in THP-1PMA cells infected or mock infected with RVFV for 2 h, followed by Z-VAD-FMK treatment with the indicted concentration for 22 h. (E) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with RVFV for the indicted times. (F) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with RVFV for 2 h, followed by Z-VAD-FMK treatment in the indicted concentration for 22 h. (G) Immunoblot analysis of caspases activation and DRP1 cleavage in THP-1PMA cells infected or mock infected with RVFV for 24 h. Stacked bar graphs show the relative levels of full-length DRP1 and cleaved DRP1 quantified by gray value analysis using ImageJ. Data are representative of three experiments with similar results. For Figure 3: Endogenous DRP1 was detected using an anti-DRP1 monoclonal antibody (CST, #5391).
Apoptotic caspase-3, −6, −7 and -8 cleave DRP1 at the AEAD556 and/or D500FAD503 motifs.
(A) Immunoblot analysis of DRP1 cleavage by the in vitro cleavage assay. The strep-tagged DRP1 protein was expressed and purified from HEK293T cells, and subjected to the in vitro cleavage assay with his-tagged caspase-1, 4, 3, 6, 7, 8 or 9 protein, followed by immunoblot analysis. Anti- strep antibody was used to determine the cleavage of DRP1. Anti-His antibody was used to identify caspases. The asterisks (*) represent non-specific bands. The blue solid rectangle represents long exposure. (B) Immunoblot analysis of whole-cell lysates (WCL, bottom) and pull-down proteins with streptavidin magnetic beads (top) from HEK293T cells transfected with indicated plasmids for 24 h. Anti-V5 antibody was used to determine DRP1. Anti-strep antibody was used to identify EGFP or caspases. (C) Prediction of the caspase cleavage sites for DRP1 using the CaspDB database. (D) Immunoblot analysis of DRP1 cleavage in Huh7 cells. Cells were transfected with the DRP1- WT or DRP1-D556E mutant expressing plasmids, followed by RVFV infection or mock infection for 24 h. (E) Immunoblot analysis of DRP1 cleavage by the in vitro cleavage assay. The strep-tagged DRP1- WT or DRP1-D556E proteins was expressed and purified from HEK293T cells, and subjected to the in vitro cleavage assay as described in (A). (F) Immunoblot analysis of DRP1 cleavage by the in vitro cleavage assay. The strep-tagged DRP1- WT or the indicated DRP1 mutants, or his-tagged caspase-3 were expressed and purified from HEK293T cells, and incubated for the in vitro cleavage assay followed by immunoblot analysis. DRP1 fragments were detected with anti-strep antibody. Caspase-3 was detected by the anti-His antibody. Data are representative of three experiments with similar results. In Figure 4, exogenous DRP1 was detected with anti-strep antibody (panels A, D, E and F) or anti-V5 antibody (panel B).
DRP1 deficiency promotes antiviral innate immunity.
(A) qPCR analysis of the indicated genes in scramble or DRP1 knockout THP-1PMA cells infected or mock infected with RVFV for 12 h. ***P < 0.001 (Two-way ANOVA test). (B) Immunoblot analysis of the indicated proteins in scramble or DRP1 knockout THP-1PMA cells infected or mock infected with RVFV for 24h. (C) Focus forming assay to determine the titer from RVFV infected scramble or DRP1 knockout THP-1PMA cells infected for 24h. ***P < 0.001 (One-way ANOVA test). (D) Immunofluorescence analysis of mitochondrial morphology in Scramble and DRP1 knockout THP-1PMA cells. Mitochondria were labeled with anti-Tom20 antibody (red), DRP1 was labeled with anti-DRP1 antibody (green, # 8570, CST) and Nuclei were stained with DAPI (blue). Scale bars, 10 μm.Data are representative of three experiments with similar results.
Caspases-mediated DRP1 cleavage promotes antiviral innate immunity.
(A) qPCR analysis of the indicated genes in vector or DRP1-WT transduced THP-1PMA cells infected or mock infected with RVFV for 12 h. ***P < 0.001 (Two-way ANOVA test). (B) Immunoblot analysis of the indicated proteins in vector or DRP1-WT transduced THP-1PMA cells infected or mock infected with RVFV for 24h. (C) Focus forming assay to determine the titer from RVFV infected vector or DRP1-WT transduced THP-1PMA cells infected with RVFV for 24h. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). (D) qPCR analysis of the indicated genes in DRP1-WT or DRP1-3DE transduced THP-1PMA cells infected or mock infected with RVFV for 12 h. ***P < 0.001 (Two-way ANOVA test). (E) Immunoblot analysis of the indicated proteins in DRP1-WT or DRP1-3DE transduced THP-1PMA cells infected or mock infected with RVFV for 24 h. (F) Focus forming assay to determine the titer from RVFV infected DRP1-WT or DRP1-3DE transduced THP-1PMA cells infected with RVFV for 24 h. **P < 0.01 (Student’s t test). (G) Immunoblot analysis of the indicated proteins in vector, DRP1-WT or DRP1-3DE transduced THP-1PMA cells infected or mock infected with RVFV for 24 h. (H) Immunofluorescence analysis of mitochondrial morphology in PCDH (control), DRP1-WT, or DRP1-3DE transduced THP-1PMA cells, infected or mock infected with RVFV for 24 h. Mitochondria were labeled with anti-Tom20 antibody (red), DRP1-WT and DRP1-3DE were labeled with anti-strep antibody (purple), RVFV were labeled with anti-RVFV-NSs antibody (green) and Nuclei were stained with DAPI (blue). Scale bars, 10 μm. Data are representative of three experiments with similar results.
Caspases-mediated DRP1 cleavage induces mitochondrial elongation to promote MAVS aggregation and immune responses.
(A) Immunofluorescence analysis of MAVS aggregation in scramble or DRP1 knockout THP-1PMA cells. Mitochondria were labeled with anti-Tom20 antibody (red), MAVS was labeled with anti- MAVS antibody (green) and Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (B) Immunoblot analysis of caspase-8 activation, DRP1 protein level and MAVS aggregation in THP-1PMA cells infected or mock infected with RVFV or 24 h. (C) Immunoblot analysis of MAVS aggregation in scramble or DRP1 knockout HEK293T cells. HEK293T cells were transfected with HA-tagged MAVS (HA-MAVS) plasmid for 24 h, followed by SeV infection for 12 h. (D) Immunoblot analysis of MAVS aggregation in scramble or DRP1 knockout THP-1PMA cells infected or mock infected with RVFV for 24 h. (E) Immunoblot analysis of the indicated proteins in DRP1 knockout or DRP1 and MAVS double knockout THP-1PMA cells infected with RVFV for 24 h. (F) Focus forming assay to determine the titer from RVFV infected DRP1 knockout or DRP1 and MAVS double knockout THP-1PMA cells infected for 24 h. ***P < 0.001 (One-way ANOVA test). Data are representative of three experiments with similar results.
Multiple viruses trigger caspases-mediated DRP1 cleavage.
(A-C) Immunoblot analysis of caspases activation and DRP1 cleavage in THP-1PMA cells infected or mock infected with H1N1 (A), SeV (B) or HSV-1 (C) for 24 h. The asterisks (*) represent non- specific bands. (D) Mitochondria were visualized by immunofluorescence using confocal microscopy. THP-1PMA cells were infected or mock infected with RVFV, H1N1, SeV or HSV-1 for 18 h, respectively. Mitochondria were labeled with anti-Tom20 (red) antibody and Nuclei were stained with DAPI (blue). Scale bars, 7.5 μm.(E) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with H1N1 for 2 h, followed by Z-VAD-FMK treatment for 22 h. (F) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with SeV for 2 h, followed by Z-VAD-FMK treatment for 10 h. (G) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with HSV- 1 for 2 h, followed by Z-VAD-FMK treatment for 22 h. (H-I) Plaque assay results determining the titers from virus infected scramble or DRP1 knockout THP-1PMA cells infected with H1N1 (H) or HSV-1 (I) for 24 h. ***P < 0.001 (One-way ANOVA test). Data are representative of three experiments with similar results.
Model of apoptotic caspases-mediated DRP1 depletion enhances anti-viral innate immunity.
Related to Figure 8. Viral infection triggers the activation of apoptotic caspases, including caspase-3, −6, −7 and 8 (C3, C6, C7 and C8). Activated apoptotic caspases cleave and inactive DRP1 at the AEAD556 and/or D500FAD503 motifs and induces mitochondrial elongation. Elongated mitochondria promote MAVS aggregation and elevate anti-viral immune responses.
RVFV infection leads to mitochondrial elongation through down-regulating DRP1.
Related to Figure 2. (A) The effects of exogenously expressed RVFV-NSs protein on mitochondrial morphology in A549 cells was observed by confocal microscopy. A549 cells were transfected with empty vector or vector expressing strep-tagged RVFV-NSs for 48 hours. Mitochondria were labeled with anti- Tom20 antibody (red), RVFV-NSs were labeled with anti-strep antibody (green) and Nuclei were stained with DAPI (blue). Scale bar, 5 μm. (B) Immunoblot analysis of DRP1 protein level in THP-1PMA cells infected with RVFV with indicated MOI. (C) Immunoblot analysis of DRP1 protein level in A549 cells infected with RVFV for 24 h. (D) Immunoblot analysis of DRP1 protein level in mouse bone marrow-derived macrophages (BMDMs) infected with RVFV for 18 h. (E) Immunoblot analysis of DRP1 protein level in the liver samples from the mice infected with RVFV at 1000 PFU for 3 days. Data are representative of three experiments with similar results.
DRP1 is cleaved by activated caspase during RVFV infection.
Related to Figure 3. (A) qPCR analysis of DRP1 mRNA in THP-1PMA cells infected with RVFV at the indicated MOI for 24 h. (B) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected or mock infected with RVFV for 24 h, or treated or untreated with Staurosporine (STS) at 1μM for 12 h. (C) Immunoblot analysis of DRP1 cleavage in THP-1PMA cells infected with RVFV at the indicated MOI. (D) Immunoblot analysis of the DRP1 cleavage in Huh7 cells transfected with empty vector or vector expressing V5-tagged DRP1 (DRP1-V5) for 24 h, followed by RVFV infection or mock infection for 24 h. The DRP1-V5 was detected by anti-V5 antibody or anti-DRP1 antibody, respectively. (E) Immunoblot analysis of the DRP1 cleavage in THP-1PMA cells, transduced with empty vector or vector expressing V5-tagged DRP1 (DRP1-V5), infected or mock infected with RVFV for 24 h. The DRP1-V5 was detected by anti-V5 antibody or anti-DRP1 antibody as indicated. (F) Immunoblot analysis of caspases activation in THP-1PMA cells infected or mock infected with RVFVWT for 24 h. (G) Immunoblot analysis of caspases activation in Huh7 cells transfected with empty vector or vector expressing the indicated proteins for 48 h. Data are representative of three experiments with similar results.
In vitro caspase activity validation and schematic of the caspase-mediated DRP1 cleavage.
Related to Figure 4. (A-C) In vitro caspase activity assays. Recombinant caspase activity was confirmed using their respective positive control substrates: (A) cGAS for caspase-1 and −4, (B) PARP1 for caspase-3, and (C) a catalytically inactive mutant of procaspase-3 (C3-C163S) for caspase-9. (D) Schematic model of caspase-mediated cleavage of the C-terminal strep-tagged DRP1, as detected by anti-strep antibody. Related to Figure 4F. (a) DRP1-WT can be cleaved by caspase-3, −6, −7 and -8 at AEAD556 motif to generate cleaved DRP1 (1) or cleaved by caspase-3 at D500FAD503 motif to generate cleaved DRP1 (2). (b) DRP1-D556E mutant can be cleaved by caspase-3 at D500FAD503 motif to generate cleaved DRP1 (2). (c-d) DRP1-D500E (c) or DRP1-D503 (d) mutant can be cleaved by caspase-3, −6, −7 and -8 at AEAD556 motif to generate cleaved DRP1 (1). (e-g) DRP1-D500/556E (e), DRP1-D503/556E (f) or DRP1-D500/503/556E (g) mutant cannot be cleaved by caspase-3, −6, −7 and -8 at AEAD556 or D500FAD503 motif to generate cleaved DRP1 (1) or cleaved DRP1 (2).
DRP1 deficiency promotes antiviral innate immunity.
Related to Figure 5. (A) Immunoblot analysis of the indicated proteins in scramble or DRP1 knockdown THP-1PMA cells infected or mock infected with RVFV for 24 h. (B) Focus forming assay to determine the titer from RVFV infected scramble or DRP1 knockdown THP-1PMA cells infected for 24 h. (C) Immunofluorescence analysis of RVFV-NSs expression in scramble or DRP1 knockout THP- 1PMA cells infected with RVFV for 24 h. Mitochondria were labeled with anti-Tom20 antibody (red), RVFV-NSs was labeled with anti-RVFV-NSs antibody (green) and Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (D) Focus forming assay to determine the titer from THP-1PMA cells infected with RVFV for 2 h, followed by mdivi-1 at the indicted concentrations for 20 h. (E) Focus forming assay to determine the titer from RVFV infected scramble or DRP1 knockout MEF cells. Cells were infected for 24 h, and cell lysate was harvested for immunoblot analysis. **P < 0.01, ***P < 0.001; ns, not significant (One-way ANOVA test). Data are representative of three experiments with similar results.
DRP1 deficiency promotes immune responses.
Related to Figure 7. (A-B) qPCR analysis of the indicted genes in scramble or DRP1 knockout THP-1PMA cells (A) or MEF cells (B). **P < 0.01, ***P < 0.001 (One-way ANOVA test). Data are representative of three experiments with similar results.
The sequences of corresponding shRNA or sgRNA sequence
