1 INTRODUCTION
Currently, a group of host cell membrane-associated proteins has been
discovered to target viral envelope glycoproteins, including IFN-induced
transmembrane (IFITM) proteins, serine incorporator (SERINC) and MARCH
proteins (1). These cellular innate immunity factors could exert their
antiviral activities at multiple stages of viral replication.
Originally, MARCH proteins were identified to show homology with the E3
ubiquitin ligase of K3 and K5 of the Kaposi’s sarcoma-associated
herpesvirus (KSHV) (2, 3). The MARCH family proteins now have been
extended to comprise at least 11 members, most of which share a similar
molecular structure, including a cytoplasmic N-terminal C4HC3
RING-finger (RING-CH finger) domain and two or more transmembrane
domains, except for MARCH7 and MARCH10, which contain no predicted
transmembrane domains. MARCH family proteins are widely involved in cell
surface protein regulation (3-9), innate immunity signal transduction
(10-12) and autophagy pathways regulation (13-15). MARCH1 and MARCH2
were originally found to downregulate the transferrin receptor (TfR) and
CD86 (B7-2) (3). MARCH1 was able to mediate MHC II ubiquitination and
thus promote dendritic cell selection of natural T regulatory cells
(16). In addition, MARCH1 could inhibit type I IFN signaling pathways
(17). MARCH2 recognized syntaxin-6 (18), regulated secretory proteins
trafficking (19), and negatively regulated cell autophagy (20) and NF-κB
essential modulator (NEMO) signaling (21).
Recently, MARCH proteins were found to show antiviral activities (22,
23). Tada et al. first reported that MARCH8 could inhibit HIV-1 and
VSV-G pseudotyped virus infection by targeting their envelope
glycoproteins (24). Subsequently, MARCH1 and MARCH2 were also found to
inhibit viral replication potentially (25-28). The viral proteins
targeted by MARCH proteins now are extended to envelope glycoproteins of
EBOV (27, 29, 30), severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) (27, 28, 30), murine
leukemia virus (MLV) (24, 27), influenza virus (IAV) (27, 29, 31), Nipah
virus (NiV) (27), spring viremia of carp virus (SVCV) (32) and rabies
virus (RABV) (28), M2 protein of IAV (33), and N protein of porcine
epidemic diarrhea virus (PEDV) (15). Interestingly, in contrast to the
negative role on viral replication, MARCH8 was also critically
supportive of hepatitis C virus (HCV) replication (34).
Generally, two antiviral modes are employed by MARCH proteins to
restrict viral replication. On one side, MARCH proteins downregulated
viral envelope glycoproteins expression and induced their intracellular
degradation, which was the viral protein c ytoplasmic t aild ependence (CTD), including envelope glycoproteins of VSV and
RABV, and M2 protein of IAV (24, 28, 33). On the other side, MARCH
proteins retained viral envelope glycoproteins at intracellular
compartments without degradation, which relied on their E3 ubiquitin
ligase activities but was the viral protein c ytoplasmict ail i ndependence (CTI), including envelope glycoproteins
of HIV-1, EBOV, and SARS-CoV-2 (24, 29, 30, 35).
EBOV GP is critical for its
target cell infection. EBOV GP0 precursor experiences
initial N-glycosylation modification in the endoplasmic reticulum (ER)
and is then transported to the trans-Golgi network (TGN), where it
finishes its mature N- and O-glycosylation (36). The mature N,
O-glycosylated GP0 is cleaved into two subunits
(GP1 and GP2) by furin protease at the
highly conserved polybasic sites (R-X-A/R-R) (36). The
GP1 and GP2 subunits are re-linked via
disulfide bonds and form GP1,2 (37), assembled into
self-trimers, and transported to the cell membrane. GP1contains a mucin-like domain (MLD), which is both N-glycosylated and
highly O-glycosylated and thus dramatically increases its molecular
weight. Therefore, the molecular weight of the mature glycosylated
GP1 is usually a little larger than the full-length
immature glycosylation form GP0.
It was previously reported that human MARCH8 retained EBOV GP at TGN and
blocked its proteolytic cleavage via targeting furin under the CTI
antiviral pattern (29). Here, we extend to investigate the anti-EBOV GP
activities of human MARCH1/2, bovine MARCH1/2, and murine MARCH1/2. Our
results show that human MARCH1 and MARCH2, bovine MARCH2, and murine
MARCH1 could inhibit EBOV GP intracellular cleavage. Critically, human
MARCH1 and MARCH2 are also found to hijack furin, as MARCH8 did, to
block EBOV GP activities, demonstrating a conserved CTI antiviral mode
among MARCH molecules and thus shedding light on subsequent antiviral
studies.