Fig 2: Interrelationship between CANX, CALR, and PDIA3 in EBOV-GP1,2 (GP) downregulation.A Expression of PDIA3, CALR, and CANX was determined in HEK293T WT, single PDIA3-, CALR-, or CANX-KO, and dual PDIA3/CALR- or PDIA3/CANX-KO cells. Protein expression was determined by WB. PDIA3, CALR, and CANX were detected by their specific antibodies. B GP was expressed with indicated ER proteins in HEK293T WT and indicated KO cells. Protein expression was detected by 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. C GP was expressed with PDIA3, CALR, or CANX in HEK293T cells. Cells were treated with indicated amounts of Tizoxanide (TIZ), and protein expression was determined by WB. GP was detected by anti-EBOV-GP; PDIA3 and CALR were detected by anti-Myc; CANX was detected by anti-HA.

Fig 3: Expression levels of the candidate proteins in the epididymis-derived exosomes and caput epididymis. A The expression of candidate proteins PDIA3, RRBP1 and CRELD2 in the exosomes detected by Western blot analysis, and their expression levels were normalized to TSG101 expression (exosome marker). All data were shown as mean ± SD, *P < 0.05, ***P < 0.001, ****P < 0.0001. B The candidate proteins PDIA3, RRBP1 and CRELD2 were widely expressed in the epithelial cells lining the lumen of caput epididymis (bar = 50 μm). C PPI showed the relationship between the candidate proteins with the other differentially expressed proteins resolved in the proteomic data. The diamonds represent the candidate proteins and the circles represent the associated proteins. The red coloration indicates the up-regulated proteins whereas the green coloration indicates the down-regulated proteins

Fig 4: 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.

Fig 5: CANX and CALR reduce EBOV entry.A HIV-1 firefly luciferase reporter viruses pseudotyped with EBOV-GP1,2 were produced from HEK293T WT, PDIA3, CALR or CANX-overexpressing, and PDIA3-, CALR- or CANX-KO cells. After infecting HEK293T cells with an equal number of these different viruses, viral entry was determined by measuring intracellular luciferase activities. Viral entry is shown as relative values, with the entry of viruses produced from HEK293T WT cells in the presence of a control vector set to 100%. B EBOV replication and transcription-competent virus-like particles (trVLPs) were produced (p0) and passaged two times (p1, p2) in HEK293T cells in the presence or absence of PDIA3, CALR, or CANX. EBOV replication was determined by measuring intracellular Renilla luciferase activity. Viral replication was also measured in the absence of EBOV-L [L(-)], which served as a negative control. Results from three independent experiments are presented. C Huh7 cells were transfected with increasing amounts of vectors expressing PDIA3, CALR, or CANX, and infected with EBOV Mayinga strain at 0.01 multiplicity of infection (MOI). Viral RNAs were extracted from the supernatants at indicated times and quantified by real-time PCR. D EBOV virus-like particles (VLPs) were produced from HEK293T WT, CALR or CANX-overexpressing, and CALR- or CANX-KO cells after expression of EBOV-GP1,2 with EBOV-VP40. VLPs were purified by ultra-centrifugation and protein expression in cell lysate and virions was analyzed by WB. GP, VP40, and endogenous CALR and CANX were detected by their specific antibodies; ectopic CALR and CANX were detected by anti-Myc or anti-HA. E Viral fusion proteins from indicated viruses were expressed with CALR in HEK293T cells (lanes 1–14). Alternatively, they were also expressed in HEK293T WT or CALR-KO cells (lanes 15-28). Protein expression was detected by WB. EBOV-GP1,2, MERS-S, SRAS1-S, and SARS2-S were detected by anti-FLAG; IAV (H5N5) HA, VSV-G, HIV-1 Env, and CALR were detected by their specific antibodies. F Viral fusion proteins from indicated viruses were expressed with CANX in HEK293T cells (lanes 1-14). Alternatively, they were also expressed in HEK293T WT or CANX-KO cells (lanes 15-28). Protein expression was detected by WB as in D, except that CANX was detected by its specific antibody. Error bars in A, B, and C represent the standard error of measurements (SEMs) calculated from three independent experiments.