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.