4 DISCUSSION
MARCH proteins have recently been shown to inhibit viral infections by
targeting their envelope glycoproteins. Generally, MARCH proteins
exerted their antiviral functions through two different modes, i.e., the
pattern of CTD or CTI. Human MARCH8 blocked EBOV GP and SARS-CoV-2 S
glycoprotein processing and glycosylation maturation through the CTI
antiviral mode (29, 30). Recent work also indiated human MARCH1, MARCH2,
and MARCH8 inhibited HIV-1, RABV, and many other viral infections
(26-28); mice MARCH1 and MARCH8 suppressed MLV and other viral infection
(27). This study further verified human MARCH1 and MARCH2, bovine
MARCH2, and murine MARCH1 suppressed EBOV GP proteolytic cleavage via
the CTI antiviral mode (Fig. 2B), demonstrating the MARCH protein
antiviral potency is conserved across many mammalian species. The
unprocessed mature glycosylation EBOV GP0 is notably
larger than GP1, as previously demonstrated (29).
However, in the presence of human MARCH1/2, bovine MARCH2, and murine
MARCH1, the mature glycosylated EBOV GP0 did not appear
(Fig. 2B), indicating these MARCHs also blocked EBOV GP glycosylation
maturation besides inhibiting its cleavage.
Like human MARCH8, human MARCH1 and MARCH2 retained EBOV GP at TGN. They
blocked its transportation to PM, thus reducing its cell surface
presentation (Fig. 3A). MARCH1 displayed a relatively high sequence
homology to MARCH8 but not MARCH2 (Fig. 1). However, they all inhibited
EBOV GP cleavage, implying protein conformation was critical to these
MARCH proteins’ CTI antiviral activities. Interestingly, both EBOV
GP0 and GP1 were devoid of expression in
the presence of bovine MARCH1 (Fig. 2B). It is uncertain which step
bovine MARCH1 blocked EBOV GP expression, i.e., at the transcription or
expression process, and further work is needed to clarify the accurate
mechanism. In contrast to its human and bovine paralogue, murine MARCH2
lost its blocking on EBOV GP cleavage (Fig. 2B), similar to a previous
report of MARCH2 inactivation in inducing MLV envelope glycoprotein
degradation (27). Given the high sequence homology between human and
murine MARCH2, it would be expected to identify which domains are
critical for their gain or loss of function in the CTI antiviral mode.
Furin cleaves a group of host intracellular proproteins, which regulate
many critical biological processes (38). It also could recognize many
viral envelope glycoproteins, including HIV-1 Env, EBOV GP, IAV H5N1 HA,
and SARS-CoV-2 S (38, 39). Viral glycoproteins cleavage by furin or
other PCs is important for their target cell infection. This study
demonstrated that human MARCH1, MARCH2, and MARCH8 specifically
interacted with furin, thus blocking EBOV GP cleavage (Fig. 4C and D).
Therefore, we deduce that other viral glycoproteins or intracellular
proproteins that critically rely on furin cleavage should also be
sensitive to human MARCH1, MARCH2, and MARCH8 mediated blocking. MARCH1,
MARCH2, and MARCH8 retained EBOV GP in TGN (Fig. 3B) and interacted with
furin and EBOV GP (Fig. 4C). These lines of evidence proved that MARCH1,
MARCH2, and MARCH8 locked the MARCH1/MARCH2/MARCH8-furin-EBOV GP complex
in TGN and thereby prevented their anterograde transport to PM, which
clarified a conserved CTI antiviral mode among these human MARCH
proteins (the antiviral mode is summarized in Fig. 6). Notably, furin is
an autocleavage protein, and mature furin is cleaved by itself at TGN or
the cell surface and then secreted outside the cells. Our results showed
that intracellular furin expression was enhanced in the presence of
human MARCH proteins (Fig. 4D, lane 3/4/5), probably because these human
MARCH proteins blocked furin autocleavage activity and retained it at
TGN, thus leading to its intracellular accumulation.
Besides MARCH8 (29), many host antiviral proteins also targeted furin to
block virus replication (40). In this report, we demonstrated that human
MARCH1 and MARCH2, and maybe bovine MARCH2 and murine MARCH1, also
inhibited furin-mediated cleavage of EBOV GP. Thus, targeting cellular
furin or other PCs to restrict enveloped viruses’ infections may be a
conserved defense mode employed by host antiviral factors and deserves
further exploration.
Generally, in pursuit of survival, viruses evolve some viral proteins to
combat host cell antiviral factors. For instance, HIV-1 Vpu and Nef
could antagonize cellular BST-2 and SERINC5, respectively (41-43);
SARS-CoV-2 ORF7a counteracted SERINC5 antiviral effects (44). Though
MARCH proteins were proven to inhibit viral replication potently, less
viral antagonizing mechanisms have been discovered.
Only recently, Liu et al. reported that the M2 protein of IAV H1N1 could
evade MARCH8 induced degradation through its cytoplasmic tail K78/K79
variation (33). Senecavirus A (SVA) 2AB was discovered to induce MARCH8
autophagic degradation (45). MARCH1, MARCH2, and MARCH8 are highly
expressed in monocyte-derived macrophages (MDM) (24, 26), which are
critical targets for EBOV infection. Thus, to effectively replicate in
MDMs, whether EBOV needs to overcome these human MARCH proteins’
restriction deserves to be investigated.