Fig 1: BILF1 triggers MAVS mitochondrial dislocation through UFMylation(A) Immunoblot of ASC oligomerization from P3HR-1 expressing the indicated sgRNA, uninduced or 4HT-induced for 24 h.(B) Immunoblot analysis of WCL from P3HR-1 expressing the indicated sgRNA and 4HT-induced, as indicated.(C) Mean ± SEM caspase-1 activity normalized by live cell number from n = 3 replicates of P3HR-1 expressing the indicated sgRNA and 4HT-treated for 24 h, as indicated.(D) Immunoblot of 1% input and anti-FLAG-MAVS complexes from 293T co-transfected with FLAG-MAVS, BILF1, or BXLF1 cDNAs for 24 h, as indicated.(E) PEAKS software identification of potential MAVS post-translational modification sites. Putative UFMylation sites were identified at lysines 362, 371, and 461. MAVS residues 321–500 are depicted. Black vertical lines represent individual peptide sequencing events.(F) Immunoblot of anti-EGFP-MAVS immunopurified from 293T co-transfected with the indicated MAVS, BILF1, and UFM1 cDNAs for 24 h.(G) Immunofluorescence analysis of wild type or K461R MAVS subcellular localization in 293T co-transfected with MAVS and BILF1 cDNAs for 24 h.(H) Immunoblot of WCL from 293T expressing MAVS and BILF1 cDNA, nigericin stimulated for 24 h, as indicated.(I) Immunoblot of 1% input and anti-HA immunopurified EGFP-MAVS and UFL1 from 293T transfected with BILF1 and EGFP-MAVS cDNAs for 24 h, as indicated.(J) AlphaFold multimer model highlighting the predicted BILF1 and MAVS interaction domain and residues.(K) Immunoblot of WCL from 293T expressing wild type, N- (?N) or C- (?C) terminal tail deletion mutant BILF1, nigericin stimulated for 24 h, as indicated.(L) Schematic of BILF1 NLRP3 inflammasome inhibition.Student’s t test was performed, with ***p < 0.001. *p < 0.05. See also Figures S4 and S5. Immunoblots are representative of n = 2.
Fig 2: MAVS sequestration and lysosomal fusion events captured in real time(A) Left: immunofluorescence analysis of MAVS and LAMP1 in P3HR-1, uninduced, or 4HT-induced for 24 h. Right: fluorescence intensity line scanning of LAMP1 (red) and MAVS (green) in the white rectangle. * and # mark co-localization and non-co-localization, respectively.(B) Mean ± SEM percentage of cells with MAVS-LAMP1 co-localization from n = 3 replicates, as in (A), using data from 12 randomly selected panels of 240 nuclei, analyzed by ImageJ ComDet plugin.(C) Immunofluorescence analysis of MAVS and LAMP1 in EBV+ AGSiZ gastric carcinoma, lytic induced by doxycycline for 24 h. Right: mean ± SEM percentage of cells with MAVS-LAMP1 co-localization from n = 3 replicates, using data from 12 randomly selected panels of 240 nuclei, analyzed by ImageJ ComDet plugin.(D) Immunoblot of 2.5% input and anti-HA immunopurified lysosomes from P3HR-1 HA-TMEM192+ cells, 4HT-induced for 24 h, as indicated. High and low molecular weight bands reactive with anti-MAVS antibodies are denoted by #. UFL1 is denoted by *. Representative of n = 2.(E) Consecutive frames captured at 10 s intervals at 3.5 h post AGSiZ lytic induction by doxycycline. White arrows highlight a GFP-MAVS puncta (green) superimposed on BFP-labeled mitochondria (blue) and SIRylo-stained lysosomes (red). Consecutive frames show partial co-localization of the GFP-MAVS puncta and lysosome (red) signals at +10 and +20 s, and then loss of MAVS puncta signal at the site of lysosome overlap at +40 s. See also corresponding Video S1.(F) Fluorescence intensity line scanning of lysosome SIRylo (red) and GFP-MAVS (green) signals at the white arrow marked puncta in (F) at +10, +20, and +40 s.(G) Schematic illustration of UFMylated MAVS trafficking from mitochondria to lysosome via MDVs.Student’s t test was performed, with ****p < 0.0001. See also Figure S6 and Video S1.
Fig 3: BILF1 mediates MAVS dislocation from the mitochondria to inhibit NLRP3 inflammasome activation(A) Immunofluorescence analysis of NLRP3 and TOMM20 in P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. Right: mean ± SEM percentage of cells with NLRP3-TOMM20 co-localization from n = 3 replicates, using data from 10 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin.(B) Immunofluorescence analysis of NLRP3 and ASC speck formation in P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h. Right: mean ± SEM percentage of cells with NLRP3/ASC specks from n = 3 replicates, using data from 20 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin.(C) Immunoblot of ASC oligomerization from P3HR-1 expressing the indicated sgRNA, uninduced, or 4HT-induced for 24 h. Representative of n = 2 replicates.(D) Immunoblot of WCL from P3HR-1 expressing the indicated sgRNA, uninduced, or 4HT-induced for 24 h. # indicates low molecular weight bands immunoreactive with anti-MAVS antibody.(E) Mean ± SEM from n = 3 replicates of caspase-1 activity normalized by live cell number from P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h.(F) Mean ± SEM from n = 3 replicates of trypan blue analysis of P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h.(G) Immunoblot of 293T transiently expressing the indicated cDNA. Representative of n = 2.(H) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. Right: mean ±SEM percentage of cells with delocalized MAVS from n = 3 replicates, using data from 20 randomly selected panels of 400 nuclei, analyzed by ImageJ ComDet plugin.(I) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1, ± BILF1 cDNA induced by 5 mM doxycycline for 24 h. Right: mean ± SEM percentage of cells with delocalized MAVS from n = 3 replicates, using data from 30 randomly selected panels of 600 nuclei, analyzed by ImageJ ComDet plugin.Student’s t test was performed, with ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. See also Figure S3.
Fig 4: BILF1 dislocates MAVS to mitochondria derived vesicles.(A) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1 expressing the indicated PARK2 or Drp1 sgRNAs and 4HT-induced for 24 h.(B) Mean ± SEM percentage of cells with dislocated MAVS from n = 3 replicates, as judged by appearance of MAVS puncta that did not overlap with TOMM20 signal as in (A), using data from 25 randomly selected panels of 500 nuclei, analyzed using ImageJ ComDet plugin.(C) Mean ± SEM percentage of P3HR-1 with NLRP3/ASC specks from n = 3 replicates, using data from 20 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin.(D) Mean ± SEM caspase-1 activity normalized by live cell number from n = 3 replicates of P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h, as indicated.(E) Immunoblot of WCL from P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h. # denotes low molecular weight bands immunoreactive with anti-MAVS antibody. Representative of n = 3.(F) PAKR2 and TOMM20 immunofluorescence analysis in 293T transfected with BILF1 or BXLF1 cDNA for 24 h.(G) Immunoblot of 1% input vs. anti-HA-MAVS immunopurified from wild type or UFL1-KO 293T transfected with MAVS and BILF1 cDNA for 24 h, as indicated. Representative of n = 2.(H) Schematic of NLRP3 inflammasome subversion by BILF1. BILF1 recruits UFL1 to mediate MAVS UFMylation, which together with PARK2 triggers selective MAVS removal from the mitochondrial outer membrane, MDV packaging and delivery to lysosomes, preventing NLRP3 inflammasome activation and pyroptosis. Student’s t test was performed, with ****p < 0.0001. ***p < 0.001. ns > 0.05. See also Figure S6.
Supplier Page from DNASU for MAVS (Homo sapiens) in pDONR221 (Gateway donor/master vector)