3.1 Human MARCH1 and MARCH2 inhibited EBOV GP proteolytic processing
Human MARCH1 and MARCH2 showed antiviral activities (26-28), and human MARCH8 could suppress EBOV GP proteolytic processing (29, 30). Thus, in this study, we aim to test whether mammalian MARCH1 and MARCH2 could block EBOV GP cleavage. Sequence alignment indicated MARCH1 shared a high, whereas MARCH2 was a low homology to MRAH8 (Fig. 1). However, they all shared a similar membrane-associated structure (23). We cloned MARCH1 and MARCH2 from humans, cattle, and mice. We then trace these mammalian MARCH proteins’ intracellular distribution. We inserted a green fluorescent protein (GFP) tag in these MARCH proteins’ C-terminus. Confocal analysis indicated that these MARCH proteins showed membrane-associated and scattered punctate distribution (Fig. 2A), similar to the intracellular localization of MARCH8 as previously reported (29). Next, we detected the anti-EBOV GP activity of these MARCH proteins. As shown in Fig. 2B, similar to humans MARCH8, MARCH1 and MARCH2 also suppressed GP processing. Similarly, bovine MARCH2 and murine MARCH1 could block GP cleavage (Fig. 2B). Intruguingly, murine MARCH2 nearly lost such inhibition effects. Notably, in the presence of bovine MARCH1, both GP0 and GP1expression was significantly impaired (Fig. 2B), which differed from the other MARCH proteins’ antiviral pattern. We then applied the GP-ΔMLD-mediated pseudovirus infection system to check whether these MARCH proteins could block viral replication (29). Compared to the vector control group, viral infectivity was significantly suppressed in the presence of the MARCH proteins except the murine MARCH2 (Fig. 2C), which matched their blocking effects on GP cleavage. Therefore, these results demonstrated that human/murine MARCH1 and human/bovine MARCH2 blocked EBOV GP proteolytic processing. In addition, human MARCH1 and MARCH2 also inhibited VSV-G pseudotyped virion infectivity (Fig. 2D) and suppressed EBOV GP cleavage in human liver Huh-7 cells (Fig. 2E).