Fig 2: 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.

Fig 3: 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 4: A model of how EBOV-GP1,2 expression is regulated in the ER via reticulorphagy.When GP1,2 levels are low in the ER, CANX-CALR cycle promotes GP1,2 folding. Properly folded GP1,2 is released from the ER to the Golgi via COPII vesicles and secreted to the plasma membrane. However, when GP1,2 levels become high in the ER, the same CANX-CALR cycle starts to trigger GP1,2 misfolding. RNF185 polyubiquitinates aberrant GP1,2 on K673 in the cytoplasmic tail via K27-linked ubiquitination. Polyubiquitinated GP1,2 is recruited to the phagophore assembly site by RETREG1 and packaged into autophagosomes via p97 ATPases and p62/SQSTM1, resulting in GP1,2 degradation in autolysosomes. Thus, CANX-CALR cycle promotes ebolavirus fitness in host cells by regulating GP1,2 expression levels during infection.

Fig 5: EBOV-GP1,2 (GP) is polyubiquitinated with K27-linked ubiquitin chains.A GP was expressed with Ub or its mutants bearing indicated single K-to-R mutation, and PDIA3, CALR, or CANX, in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. B GP was expressed with Ub or its mutants expressing indicated single lysine residue, and PDIA3, CALR, or CANX, in HEK293T cells. GP proteins were immunoprecipitated and GP polyubiquitination was analyzed by WB. In A and B, GP was detected by anti-EBOV-GP; PDIA3, CALR, and CANX were detected by anti-Myc; Ub was detected by anti-HA.