3.3 MARCH1 and MARCH2 inhibited furin-mediated cleavage of EBOV
GP
MARCH8 suppressed furin-mediated cleavage of EBOV GP (29). Therefore,
based on the above results (Fig. 2B), we further verified whether MARCH1
and MARCH2 could block furin-mediated GP cleavage. We first applied the
BiFC system to detect MARCH1/MARCH2/MARCH8 and furin intracellular
interaction. MARCH1-VN/MARCH2-VN/MARCH8-VN and furin-VC (FR-VC) were
co-transfected into 293T cells. Flow cytometry analysis showed
MARCH1-VN/MARCH2-VN/MARCH8-VN, and FR-VC had strong intracellular
interaction signals (Fig. 4A). Then, MARCH1-VN/MARCH2-VN/MARCH8-VN and
FR-VC were co-transfected into HeLa cells. As indicated in Fig. 4B,
these MARCH proteins and furin had a clear intracellular
co-localization. Next, to confirm their intracellular interactions, the
Co-IP assay was performed. The furin expression vector was
co-transfected with MARCH1/MARCH2 and EBOV GP-ΔMLD expression vectors to
further verify furin’s binding to MARCH proteins and EBOV GP. As shown
in Fig. 4C, MARCH1/2, and GP-ΔMLD could be co-immunoprecipitated by
furin, indicating they formed an intracellular complex, similar to the
results obtained in MARCH8, as previously identified (29). Relying on
their intracellular interactions, we identify whether these MARCH
proteins blocked furin-mediated GP cleavage. Generally, mature
GP0 was cleaved into GP1(~130 kDa) and GP2 (~26
kDa) by endogenous furin, whereas in the presence of exogenous furin
overexpression, mature GP0 was cleaved into three parts,
i.e., the GP1, GP1*and GP2 (Fig. 4D, lane 2), as previously demonstrated
(29). Full-length GP was co-transfected with furin in the absence or
presence of these MARCH proteins. In the presence of MARCH1, MARCH2, or
MARCH8, furin-mediated GP cleavage was inhibited because very few
GP1, GP1*, and
GP2 were produced (Fig. 4D, lane 3/4/5). Thus, these
results demonstrated MARCH1, MARCH2, and MARCH8 could inhibit
furin-mediated GP cleavage.