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.