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).