Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop

Murin, Charles D.; Bruhn, Jessica F.; Bornholdt, Zachary A.; Copps, Jeffrey; Stanfield, Robyn L.; Ward, Andrew B.

doi:10.1016/j.celrep.2018.08.009

Cited by 27 publications

(31 citation statements)

References 67 publications

(108 reference statements)

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“…In contrast, ADI-15946, an antibody that competes with KZ52 but bridges the GP1 core and glycan cap [33][34][35], modestly stabilized the high-FRET state. We also tested nAbs ADI-15878 and ADI-15742, which contact the GP2 fusion loop and adjacent GP1 protomer [33,34,36]. These nAbs significantly increased the occupancy in the high-FRET state (Figure 5b).…”

Section: Neutralizing Antibodies Mediate Gp Conformationmentioning

confidence: 99%

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Durham

Howard

Govindan

et al. 2020

Viruses

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The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

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Section: Neutralizing Antibodies Mediate Gp Conformationmentioning

confidence: 99%

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Durham

Howard

Govindan

et al. 2020

Viruses

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“…Engineered glycoprotein trimers from other enveloped viruses (e.g., Middle East respiratory syndrome coronavirus, MERS-CoV, and parainfluenza virus types 1-4) were designed to present the antigenically optimal prefusion conformation Stewart-Jones et al 2018). Glycoproteins from Ebola virus were modified by removing the mucin-like domain, assembled as soluble trimers, and then studied in a complex with the ADI-15878 Fab by SPA (Murin et al 2018). Except for the above-mentioned trimeric forms, glycoprotein complexes containing more than one viral glycoprotein (e.g., gH/gL/gp42 from Epstein-Barr virus) have also been used for cryo-EM antibody-antigen studies (Snijder et al 2018).…”

Section: Enveloped Viruses Without Icosahedral Symmetrymentioning

confidence: 99%

Cryo-EM Studies of Virus-Antibody Immune Complexes

et al. 2020

Virol. Sin.

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Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.

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“…Because GP→GP CL cleavage is a prerequisite for cell entry by all filoviruses that have been evaluated to date (22,24,47), we postulated that this cleavage also destabilizes GPs from divergent ebolaviruses. Accordingly, we used the pan-ebolavirus base-binding mAb ADI-15878 to probe the thermal stability of GPs from the divergent ebolaviruses Sudan virus (SUDV) and the recently discovered Bombali virus (BOMV) in the epitope-loss ELISA (48)(49)(50)(51). THL cleavage of SUDV GP and BOMV GP was verified by western blot ( Fig 3A).…”

Section: The Gp Proteins Of Other Ebolaviruses Are Also Destabilized mentioning

confidence: 99%

A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition

Bortz

Wong

Grodus

et al. 2020

Preprint

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Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases.Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes . The temperature-dependent stability (thermostability) of the pre-fusion conformers of 'Class I' viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple ELISA-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of pre-fusion conformation at elevated temperatures, but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP CL . Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals. ImportanceThough a vaccine for Ebola virus has been approved by the FDA within the past year, no FDA-approved therapeutics are available to treat infections by Ebola virus or other filoviruses. The development of such countermeasures is challenged by our limited understanding of the mechanism by which Ebola virus enters cells, especially at the final step of membrane fusion. The sole surface-exposed viral protein, GP, mediates key steps in virus entry, including membrane fusion, and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these structural rearrangements.Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer forms of GP with enhanced stability that cou...

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Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop

Cited by 27 publications

References 67 publications

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Cryo-EM Studies of Virus-Antibody Immune Complexes

A virion-based assay for glycoprotein thermostability reveals key determinants of filovirus entry and its inhibition

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