Structures of Marburgvirus glycoprotein and its complex with NPC1 receptor | Nature

Download PDF Subjects Cryoelectron microscopy Marburg virus Abstract Marburgviruses (MBVs) cause severe haemorrhagic fever with higher fatality rates than Ebola virus (EBOV) 1 , 2 , 3 , 4 . Here we show that the MBV glycoprotein (GP) mediates viral entry more efficiently than EBOV GP. Using cryo-EM, we determined structures of MBV GP in three states: (1) unbound; (2) bound to its endosomal receptor NPC1; and (3) complexed with a neutralizing nanobody. The glycan cap shields the receptor-binding site from NPC1 but only partially from the nanobody, enabling limited immune evasion. After glycan cap cleavage, NPC1 binds to MBV GP in a distinct orientation compared with EBOV GP, providing an additional anchor and enhancing receptor affinity. NPC1 engagement also induces substantial conformational changes in MBV GP, probably facilitating membrane fusion. Furthermore, MBV GP is susceptible to the neutralizing nanobody, which mimics NPC1 at the receptor-binding site. Together, our findings reveal MBV GP as a highly efficient entry mediator and suggest structural mechanisms that may contribute to its enhanced entry efficiency. Main MBVs, members of the Marburgvirus genus within the Filoviridae family, cause severe haemorrhagic fever in humans and non-human primates 3 , 4 . Since their discovery in 1967, 18 MBV outbreaks have been reported, most recently in 2024 1 . The two known MBV species, Marburg virus (MARV) and Ravn virus (RAVV), are closely related and often not distinguished in outbreak reports. MBVs have been isolated from bats 5 , confirming bats as their natural reservoir and suggesting long-term co-existence with human populations. The average case fatality rate of MBV infection is 73% (409 deaths among 563 reported human cases), markedly higher than that of EBOV, a member of the Ebolavirus genus, which averages 44% (14,881 deaths among 33,820 cases) 1 , 2 . Although two antibody-based therapies and one vaccine have been approved for EBOV 6 , 7 , no licensed therapeutics or vaccines exist for MBVs. Understanding the causes of the high lethality of MBV and developing effective countermeasures are critical for pandemic preparedness and global health security. Viral entry into host cells is a key determinant of infectivity and pathogenesis, and a major target for neutralizing antibodies 8 , 9 . The EBOV GP, which mediates entry, has been extensively studied 10 , 11 . On the viral surface, GP exists in a trimeric pre-fusion state composed of three copies each of the receptor-binding subunit GP1 and the membrane-fusion subunit GP2 (ref. 12 ). A defining feature of EBOV GP is that its receptor-binding site (RBS) is shielded by a glycan cap and a mucin-like domain (MLD), blocking antibody access and promoting immune evasion 13 . During entry, GP1 engages host factors at the cell surface to trigger endocytosis 14 . Inside the endosome, proteases remove the glycan cap and MLD 15 , exposing the RBS and generating a cleaved form of GP (GPcl) that binds its receptor, Niemann–Pick C1 (NPC1) 10 , 11 , 16 . The crystal structure of EBOV GPcl bound to domain C of NPC1 (NPC1-C) shows that NPC1-C inserts two loops into a hydrophobic cavity at the top of GPcl to engage the RBS 10 . NPC1 binding then drives GP2 to transition into its post-fusion conformation, mediating membrane fusion 17 . Thus, EBOV entry depends on three essential steps: glycan cap removal, NPC1 binding and NPC1-induced conformational rearrangements of GP. Compared with EBOV GP, MBV GP is much less characterized. Like EBOV, MBV GP uses NPC1 as its endosomal receptor 18 . To date, only two crystal structures of MBV GP have been reported, both in complex with neutralizing antibodies 13 , 19 . The structures of MBV GP alone or bound to NPC1 remain unknown, leaving major gaps in our understanding of its function. Neither the binding affinity of MBV GP for NPC1 nor the structural changes induced by NPC1 have, to our knowledge, yet been defined. Unlike EBOV GP, where the RBS is shielded, neutralizing antibodies can directly target the RBS of MBV GP 13 , 19 . This observation has led to the suggestion that the MBV GP glycan cap is flexible, permitting antibody access and challenging its presumed role in immune evasion 13 , 19 . Of note, the efficiency of MBV GP in mediating viral entry has not been investigated, despite its likely contribution to the high lethality of MBV infections. In this study, we compared the entry efficiencies mediated by MBV and EBOV GPs. We determined cryo-electron microscopy (cryo-EM) structures of MBV GP in three distinct states: (1) unbound, (2) bound to human NPC1-C, (3) and bound to a neutralizing nanobody (a single-domain antibody from camelids 20 , 21 , 22 , 23 , 24 ). To complement these structural analyses, we performed biochemical assays. Together, our findings establish MBV GP as a highly efficient mediator of viral entry and provide new structural insights into its receptor recognition, cell entry and immune evasion, while als