3.2 Human MARCH1 and MARCH2 retained EBOV GP in the TGN
apparatus.
Generally, mature GP1,2 is presented on the cell
surface, enveloped into virions, and responsible for binding target
cells’ receptors in EBOV next round of infection. Human MARCH1 and
MARCH2 reduced both GP1 expression and
GP1,2-mediated viral infectivity. We thus deduced that
human MARCH1 and MARCH2 downregulated GP1 cell surface
display. To confirm the hypothesis, the GP-ΔMLD-GFP was co-transfected
with human MARCH1 or MARCH2. GP-ΔMLD-GFP displayed a
plasma membrane (PM)
localization, and the human MARCH1 and MARCH2 proteins showed scattered
punctate distribution (Fig. 3A). In the presence of human MARCH1 or
MARCH2, the GP-ΔMLD-GFP protein was downregulated from PM and was
retained intracellularly (Fig. 3A). This evidence demonstrated MARCH1
and MARCH2 trapped GP-ΔMLD-GFP at an intracellular compartment, where it
was unable to be transported to PM. To further verify this, we
introduced the bimolecular fluorescence complementation (BiFC) system to
determine where the GP was retained. MARCH proteins were fused with the
Venus N-terminus (VN, residues 2-173) in their C-terminus, while the
GP-ΔMLD was fused with the Venus C-terminus (VC, residues 154-238) in
its C-terminus. Simultaneously, we applied the calnexin-mCherry
(CNX-mCherry) and TGN46-mCherry (TGN-mCherry) as the ER and TGN
localization markers, respectively (29). MARCH1-VN/MARCH2-VN/MARCH8-VN
and GP-ΔMLD-VC were co-transfected with CNX-mCherry or TGN-mCherry to
trace their intracellular localization. Individual
MARCH1-VN/MARCH2-VN/MARCH8-VN/GP-ΔMLD-VC was negative for any
fluorescent signals (data not shown). In the presence of MARCH1/2/8,
EBOV GP-ΔMLD was trapped intracellularly, consistent with the former
results (Fig. 3B). Further detection indicated
MARCH1-VN/MARCH2-VN/MARCH8-VN and GP-ΔMLD-VC pair groups showed minimal
overlap with CNX-mCherry but displayed a strong co-localization with
TGN-mCherry (Fig. 3B). Therefore, we concluded MARCH1 and MARCH2
retained GP in TGN, as MARCH8 did.