FIGURE LEGENDS
Figure 1. Amino acid (aa) sequence alignment of MARCH proteins.Human MARCH8 aa sequence was aligned with MARCH1 and MARCH2. Dots represent identical aa, while dishes indicate the deletion. M1, human MARCH1; M2, human MARCH2; M8, human MARCH8.
Figure 2. MARCH proteins blocked EBOV GP cleavage. A. human/bovine/murine MARCH1/2-GFP and human MARCH8-GFP vectors were transfected into HeLa cells. Nuclei were stained with DAPI, and fluorescent signals were detected by confocal microscopy. B. GP (full-length) was co-expressed with human/bovine/murine MARCH1/2 or human MARCH8 in 293T cells. GP was detected with anti-FLAG, and MARCH proteins were detected with anti-HA. C. GPΔMLD, pNL-Luc-ΔEnv, and MARCH expression vectors were co-transfected into 293T cells. Supernatants containing the pseudovirions were collected to infect Vero cells. Infectivity was indicated as luciferase values. D. VSV-G, pNL-Luc-ΔEnv, and human MARCH1/2 expression vectors were co-transfected into 293T cells. Supernatants containing the pseudovirions were collected to infect Vero cells. E. GP (full-length) was co-expressed with human MARCH1/2 in human Huh-7 cells. GP was detected with anti-FLAG, and MARCH proteins were detected with anti-HA. HM, human MARCH; BM, bovine MARCH; MM, murine MARCH; M1, human MARCH1; M2, human MARCH2.
Figure 3. Intracellular co-localization assay for MARCH proteins and EBOV GP. A. MARCH1/2 was co-expressed with the GPΔMLD-GFP or empty vector in HeLa cells. GPΔMLD-GFP was used as a control. MARCH1/2 was detected by anti-HA, and nuclei were stained with DAPI. Fluorescent signals were monitored by confocal microscopy. B. In HeLa cells, the MARCH1/2/8-VN/GPΔMLD-VC BiFC pairs were co-expressed with CNX-mCherry or TGN-mCherry, and nuclei were stained with DAPI. Fluorescent signals were collected through confocal microscopy. M1, human MARCH1; M2, human MARCH2; M8, human MARCH8; TGN-MC, TGN-mCherry; CNX-MC, CNX-mCherry.
Figure 4. MARCH proteins suppressed EBOV GP intracellular cleavage by furin. A. MARCH1/2/8-VN/furin-VC BiFC pairs were co-transfected into 293T cells. After 36 h, cell samples were collected and pretreated for flow cytometry assay. MARCH8-VN was previously indicated negative for any fluorescent signals (29). B. MARCH1/2/8-VN/furin-VC BiFC pairs were co-transfected into HeLa cells. The MARCH1/2/8-VN proteins were fused with a C-terminal HA tag. Cells were stained with fluorescent anti-HA to detect MARCH proteins and with DAPI to detect nuclei. Fluorescent signals were obtained under confocal microscopy. C. FLAG-tagged furin was co-expressed with HA-tagged MARCH1/2 and HA-tagged GPΔMLD proteins in 293T cells. Samples were immunoprecipitated with anti-FLAG and used for WB assay. Furin was detected with anti-FLAG, and MARCH1/2 and GPΔMLD were detected by anti-HA. D. GP was co-expressed with furin and MARCH1/2/8. GP and furin were detected by anti-FLAG, and MARCH1/2/8 were detected with anti-HA. FR, furin; M1, human MARCH1; M2, human MARCH2; M8 human MARCH8; SP, signal peptide.
Figure 5. Critical domain identification of furin interacted with human MARCH1/2/8. Full-length or Flag-tagged ΔCD/ΔP furin was co-immunoprecipitated with HA-tagged MARCH1/2/8. Furin was detected with anti-FLAG, and MARCH1/2/8 were detected with anti-HA. M1, human MARCH1; M2, human MARCH2; M8 human MARCH8; ΔCD/ΔP, furin ΔCD/ΔP mutant.
Figure 6. Antiviral model of MARCH molecules in inhibiting EBOV GP maturation. MARCH proteins were localized in membrane-associated compartments, including ER, TGN, endosomes, and PM. EBOV GP experiences mature glycosylation modification at TGN and is cleaved by Furin into the GP1 and GP2 subunits. The GP1 and GP2 are re-linked and transported from TGN to PM. However, in the presence of MARCH proteins, furin interacted with both MARCHs and EBOV GP and formed the MARCH-furin-EBOV GP complex at TGN. Resultantly, EBOV GP cleavage/glycosylation maturation was blocked and was trapped by MARCH proteins at TGN, which blocked its translocation from TGN to PM. PM, plasma membrane; TGN, trans-Golgi network; ER, endoplasmic reticulum.