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.