Fig 2: Palmitic acid (PA) significantly induces the ER stress in the primary EECs and promotes the EEC-derived exosome biogenesis in EECs. A Co-immunofluorescent staining of cytokeratin 8 (red) and vimentin (green) evaluated primary EECs purity. B-D PA significantly induced ER stress based on the upregulation of PERK and IRE1α in the EECs. E-F Western blot analysis detected the expression levels of the candidate proteins, PDIA3 and RRBP1, in the EECs with or without PA treatment. G-H Representative Western blot analyses of candidate protein CRELD2 and exosome marker CD63 in the EECs with or without PA treatment. I NTA determination of the concentration of the exosomes derived from the EECs. The relative quantification of the proteins was normalized to β-actin. All the data were presented as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001

Fig 3: RNF185 polyubiquitinates and degrades EBOV-GP1,2 (GP).A GP was expressed with His-tagged Ub in the presence of indicated E3 Ub ligases or their catalytically inactive mutants in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP was expressed with indicated proteins in HEK293T cells. Protein expression was determined by WB. GP, RNF26, RNF185, TRIM25, and MARCH8 were detected as in A; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA. C GP was expressed with RNF185 or RNF26 and treated with indicated inhibitors as we did previously. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF26 and RNF185 proteins were detected by anti-FLAG. D A schematic diagram of RNF185 is presented. Two transmembrane (TM1, TM2) domains and a RING-finger are shown. Indicated RNF26 mutants were constructed. E GP was expressed with RNF185 and its mutants in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG. F GP was expressed with GFP, RNF185, and indicated RNF185 mutants in HEK293T cells. GFP and RNF185 proteins were immunoprecipitated and their interaction with GP was analyzed by WB. GP was detected by anti-EBOV-GP; GFP and RNF185 proteins were detected by anti-FLAG. G GP was expressed with Ub with a His-tag and RNF185 or its indicated mutants in HEK293T cells. GP and RNF185 proteins were immunoprecipitated and GP polyubiquitination was determined by WB. GP was detected by anti-EBOV-GP; RNF185 proteins were detected by anti-FLAG; Ub was detected by anti-His.

Fig 4: CANX and CALR decrease ebolavirus GP1,2 expression at steady state.A EBOV-GP1,2 (GP) was expressed with indicated ER proteins in HEK293T cells. Protein expression was detected by western blotting (WB). GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; HSPA5 was detected by anti-FLAG; CANX was detected by anti-HA. Ctrl, control vector; ACTB, β-actin. B Indicated EBOV glycoproteins were expressed with CALR or CANX in HEK293T cells. Protein expression was detected by WB. EBOV glycoproteins were detected by anti-HiBiT; CALR was detected by anti-Myc; CANX was detected by anti-HA. C Indicated EBOV glycoproteins were expressed with CANX or CALR in HEK293T cells, and co-immunoprecipitated (IP) with anti-EBOV-GP from cell lysate (Input). The GP interactions with CANX and CALR were determined by WB. GPs were detected by anti-EBOV-GP; CALR was detected by anti-Myc; CANX was detected by anti-HA. D EBOV-GP1,2 was expressed in HEK293T wild-type (WT) and PDIA3-, CALR-, or CANX-knockout (KO) cells. Protein expression was detected by WB. GP and endogenous PDIA3, CALR, and CANX were detected by their specific antibodies. E EBOV-GP1,2 was expressed with increased amounts of CALR or CANX in HEK293T WT and CALR- or CANX-KO cells. Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA. F GP1,2 proteins from indicated ebolaviruses were expressed with CALR or CANX in HEK293T cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; CALR and CANX were determined by anti-Myc or anti-HA. G GP1,2 proteins from indicated ebolaviruses were expressed in HEK293T WT and CALR- or CANX-KO cells. Protein expression was detected by WB. GPs were detected by anti-EBOV-GP; CALR and CANX were detected by their specific antibodies. H MARV-GP1,2 was expressed with CANX or CALR in HEK293T cells. Alternatively, MARV-GP1,2 was expressed in HEK293T WT and CALR- or CANX-KO cells. Protein expression was detected by WB. MARV-GP was detected by anti-HiBiT; ectopic CALR and CANX were detected by anti-Myc or anti-HA; endogenous CALR and CANX were detected by their specific antibodies. I EBOV-GP1,2 was expressed with CANX or CALR in A549 cells, HeLa cells, Hep G2 cells, primary mouse macrophages (Mϕ), and THP1-derived macrophages (THP1-Mϕ). Protein expression was detected by WB. GP was detected by anti-EBOV-GP; CALR and CANX were detected by anti-Myc or anti-HA.

Fig 5: RNF185 polyubiquitinates EBOV-GP1,2 (GP) on K673 in the cytoplasmic tail.A GP and its mutants K673A or ∆CT were expressed with indicated E3 Ub ligases in HEK293T cells. GP and E3 proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; MARCH8 was detected by anti-HA; Ub was detected by anti-His. B GP and its two mutants were expressed with His-tagged Ub in the presence of PDIA3, CALR, or CANX in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA; Ub was detected by anti-His. C GP and its two mutants were expressed with indicated E3 ubiquitin ligases or ER proteins in HEK293T cells. Protein expression was determined by WB. GP was detected by anti-EBOV-GP; MARCH8 and CANX were detected by anti-HA; RNF26, RNF185, and TRIM25 were detected by anti-FLAG; PDIA3 and CALR were detected by anti-Myc. D RNF185 was expressed with a control (Ctrl) or RNF185-specific siRNAs in HEK293 cells. RNF185 expression was determined by WB using anti-FLAG. E GP was expressed with PDIA3, CALR, or CANX in the presence of Ctrl or RNF185-specific siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP. F GP was expressed with CALR, CANX, or PDIA3, and indicated siRNAs in HEK293T cells. GP expression was determined by WB using anti-EBOV-GP.