Figure 7: The effect of mitochondrial-targeted PB1-F2 in antiviral innate immunity.

(a,b) HEK293 cells were co-transfected with 75 ng of plasmid encoding Myc-tagged MAVS, increasing amounts (20, 50 and 100 ng) of plasmids encoding the PB1-F2 variants, and either the (a) IFN-β or the (b) NF-κB reporter plasmids. Transfected cells were analyzed 24 h later for reporter gene-dependent luciferase activities. In these assays, we used Su9-eGFP plasmids as the control. Western blots reveal the abundance of Myc-MAVS proteins. (c) HEK293 cells were co-transfected with 75 ng of plasmid encoding Myc-tagged RIG-I (1-250), 100 ng of PB1-F2 chimeric mutants, and IFN-β reporter plasmids. (d) HEK293 cells were co-transfected with 30 ng of plasmid encoding NLRP3, ASC (5 ng), procaspase-1 (5 ng), pro-IL-1β (150 ng) and each PB1-F2 variant (300 ng). Cell-free supernatants were collected 24 h post transfection, and secreted IL-1β was measured by ELISA. In these assays, we used the eGFP plasmid as the control. Inset: the functional role of the MOM-localized PB1-F2 chimeric mutant in NLRP3 inflammasomes. (e) The reconstituted level of secreted IL-1β in d was confirmed by immunoblotting with antibodies against human IL-1β and pro-IL-1β, and β-actin was used as a loading control in whole cell lysates. (f) The inhibition of ASC oligomerization by mitochondrial-targeted PB1-F2 variants. HEK293 cells transfected with plasmids encoding NLRP3, FLAG-tagged ASC, and each PB1-F2 variant were lysed and their lysates were cross-linked with BS3. The samples were analyzed by immunoblotting with antibody against FLAG. The bottom blots confirm the expression levels of NLRP3 and ASC proteins in their lysates. All data represent the mean values±s.d. (n=3 experiments). N.S., not significant, *P<0.05, **P<0.01, and ***P<0.001 (by unpaired t-test), respectively.