Fig 1: SFTSV NSs interacts with CARD8.(A) Colocalization of NSs, NSs-8A and NLRP1 in HEK293T cells co-transfected with HA-NSs, HA-NSs-8A and V5-NLRP1 for 48 h. Scale bar is 10 μm. (B) Co-IP assay between HA-NSs and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (C) Co-IP assay between HA-NSs and Flag-CARD8-FIIND in HEK293T cells transfected with the indicated expression vectors for 48 h. (D) Co-IP assay between Flag-NSs or Flag-NSs-8A and Myc-CARD8 in HEK293T cells transfected with the indicated expression vectors for 48 h. (E) GSDMD cleavage in THP-1 cells infected with lentiviruses expression Flag-NSs-WT or Flag-NSs-8A or carrying vector for 48 h.
Fig 2: The role of SFTSV NSs in the activation of the NLRP1 and CARD8 inflammasome.(A) ASC-caspase-1-pro-IL-1β HEK293T cells were transfected with indicated expression vector for 36 h. Supernatants were analyzed for IL-1β with ELISA. (B) ASC-caspase-1-pro-IL-1β HEK293T cells were transfected with plasmids NSs, production of p17 and GSDMD processing were detected with Western blot. (C-D) ASC specks fluorescence microscopy images (C) and quantification (D) of A549-HA-NLRP1-Flag-ASC-GFP cells transfected NSs-HA for 24 h. Scale bar, 100 μm. (E-F) GSDMD cleavage (E) and IL-1β (F) production in primary keratinocytes expressing Tet-HA-NSs and treated with doxycycline (1 μg/ml) for 48 h. (G-H) GSDMD cleavage (G) and IL-1β (H) production in THP-1 cells expressing Tet-HA-NSs and treated with doxycycline (1 μg/ml) for 48 h. All data represent three independent experiments and presented as mean±s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. For statistical analysis, two-tailed unpaired Student’s t-test in (A, D, F, H).
Fig 3: SFTSV activates the CARD8 inflammasome in macrophages.(A) GSDMD cleavage in THP-1 cells infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h. (B) Knockout of CARD8 in THP-1 cells identified with Western blot. (C) Knockout of NLRP3 in THP-1 cells identified with Western blot. (D-F) GSDMD cleavage (D), IL-1β production (E) and LDH release (F) in THP-1 WT, CARD8 KO and NLRP3 KO infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 24 h. (G) THP-1 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (H) A549 cells were infected with SFTSV at different MOIs for 24 h, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. (I) A549 cells were infected with SFTSV (MOI = 1) at indicated time, endogenous CARD8 was detected using N-terminal and C-terminal antibodies with Western blot, respectively. All data represent three independent experiments and presented as mean±s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. For statistical analysis, two-tailed unpaired Student’s t-test in (E, F).
Fig 4: NSs impairs the interaction of NLRP1/CARD8 and DPP9.(A) Primary keratinocytes were infected with SFTSV (MOI = 1) or stimulated with VbP (5 μM) for 36 h, endogenous DPP8 and DPP9 were detected with Western blot. (B) THP-1 cells were infected with SFTSV (MOI = 1) or stimulated with VbP (2 μM) for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (C) A549 cells were infected with different MOI SFTSV for 24 h, endogenous DPP8 and DPP9 were detected with Western blot. (D) A549 cells expressing Tet-HA-NSs were treated with doxycycline (1 μg/ml) for 48 h, and identified with Western blot. (E) A549-Tet-HA-NSs cells were treated with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (F) THP-1-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h, endogenous DPP8 and DPP9 were detected with Western blot. (G) A549-Tet-HA-NSs cells were treatment with doxycycline (1 μg/ml) for 48 h and then treated with MG132 (10 μM) or CQ (50 μM) for 6 h before harvest. (H) Co-IP of NLRP1-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (I) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with indicated expression vectors for 48 h. (J) Co-IP of CARD8-DPP9 with NSs in HEK293T cells transfected with Myc-CARD8 and HA-NSs for 48 h.
Fig 5: CARD8 deletion promotes SFTSV replication.(A-D) THP-1-Ctl, CARD8 KO, and NLRP3 KO were infected with SFTSV (MOI = 1) for 48 h, the mRNA levels (A) of SFSTV S, M, L segments were detected with RT-qPCR; (B) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm; (C) Expression of SFTSV NP was analyzed with Western blot; (D) Functional titers of SFTSV in Ctl, CARD8-KO and NLRP3-KO THP-1 cells measured by TCID50. (E-F) THP-1 cells were infected with SFTSV (MOI = 1) and treated with VbP (2 μM) for 24 h. (E) Expression of SFTSV NP was analyzed with Western blot; (F) cells were stained with SFTSV NP and analyzed with immunofluorescence assays. Scale bar, 100 μm. All data represent three independent experiments and presented as mean±s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. For statistical analysis, two-tailed unpaired Student’s t-test in (A, D).
Supplier Page from Sino Biological, Inc. for Human CARD8 Gene ORF cDNA clone expression plasmid, N-Myc tag