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.