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.